Vitronectin receptor antagonist pharmaceuticals

ABSTRACT

The present invention describes novel compounds of the formula:  
     (Q) d -L n -C h ,  
     useful for the diagnosis and treatment of cancer, methods of imaging tumors in a patient, and methods of treating cancer in a patient. The present invention also provides novel compounds useful for monitoring therapeutic angiogenesis treatment and destruction of new angiogenic vasculature. The present invention further provides novel compounds useful for imaging atherosclerosis, restenosis, cardiac ischemia and myocardial reperfusion injury. The present invention still further provides novel compounds useful for the treatment of rheumatoid arthritis. The pharmaceuticals are comprised of a targeting moiety that binds to a receptor that is upregulated during angiogenesis, an optional linking group, and a therapeutically effective radioisotope or diagnostically effective imageable moiety. The imageable moiety is a gamma ray or positron emitting radioisotope, a magnetic resonance imaging contrast agent, an X-ray contrast agent, or an ultrasound contrast agent.

FIELD OF THE INVENTION

[0001] The present invention provides novel pharmaceuticals useful forthe diagnosis and treatment of cancer, methods of imaging tumors in apatient, and methods of treating cancer in a patient. Thepharmaceuticals are comprised of a targeting moiety that binds to thevitronectin receptor that is expressed in tumor vasculature, an optionallinking group, and a therapeutically effective radioisotope ordiagnostically effective imageable moiety. The therapeutically effectiveradioisotope emits a gamma ray or alpha particle sufficient to becytotoxic. The imageable moiety is a gamma ray or positron emittingradioisotope, a magnetic resonance imaging contrast agent, an X-raycontrast agent, or an ultrasound contrast agent.

BACKGROUND OF THE INVENTION

[0002] Cancer is a major public health concern in the United States andaround the world. It is estimated that over 1 million new cases ofinvasive cancer will be diagnosed in the United States in 1998. The mostprevalent forms of the disease are solid tumors of the lung, breast,prostate, colon and rectum. Cancer is typically diagnosed by acombination of in vitro tests and imaging procedures. The imagingprocedures include X-ray computed tomography, magnetic resonanceimaging, ultrasound imaging and radionuclide scintigraphy. Frequently, acontrast agent is administered to the patient to enhance the imageobtained by X-ray CT, MRI and ultrasound, and the administration of aradiopharmaceutical that localizes in tumors is required forradionuclide scintigraphy.

[0003] Treatment of cancer typically involves the use of external beamradiation therapy and chemotherapy, either alone or in combination,depending on the type and extent of the disease. A number ofchemotherapeutic agents are available, but generally they all sufferfrom a lack of specificity for tumors versus normal tissues, resultingin considerable side-effects. The effectiveness of these treatmentmodalities is also limited, as evidenced by the high mortality rates fora number of cancer types, especially the more prevalent solid tumordiseases. More effective and specific treatment means continue to beneeded.

[0004] Despite the variety of imaging procedures available for thediagnosis of cancer, there remains a need for improved methods. Inparticular, methods that can better differentiate between cancer andother pathologic conditions or benign physiologic abnormalities areneeded. One means of achieving this desired improvement would be toadminister to the patient a metallopharmaceutical that localizesspecifically in the tumor by binding to a receptor expressed only intumors or expressed to a significantly greater extent in tumors than inother tissue. The location of the metallopharmaceutical could then bedetected externally either by its imageable emission in the case ofcertain radiopharmaceuticals or by its effect on the relaxation rate ofwater in the immediate vicinity in the case of magnetic resonanceimaging contrast agents.

[0005] This tumor specific metallopharmaceutical approach can also beused for the treatment of cancer when the metallopharmaceutical iscomprised of a particle emitting radioisotope. The radioactive decay ofthe isotope at the site of the tumor results in sufficient ionizingradiation to be toxic to the tumor cells. The specificity of thisapproach for tumors minimizes the amount of normal tissue that isexposed to the cytotoxic agent and thus may provide more effectivetreatment with fewer side-effects.

[0006] Previous efforts to achieve these desired improvements in cancerimaging and treatment have centered on the use of radionuclide labeledmonoclonal antibodies, antibody fragments and other proteins orpolypeptides that bind to tumor cell surface receptors. The specificityof these radiopharmaceuticals is frequently very high, but they sufferfrom several disadvantages. First, because of their high molecularweight, they are generally cleared from the blood stream very slowly,resulting in a prolonged blood background in the images. Also, due totheir molecular weight they do not extravasate readily at the site ofthe tumor and then only slowly diffuse through the extravascular spaceto the tumor cell surface. This results in a very limited amount of theradiopharmaceutical reaching the receptors and thus very low signalintensity in imaging and insufficient cytotoxic effect for treatment.

[0007] Alternative approaches to cancer imaging and therapy haveinvolved the use of small molecules, such as peptides, that bind totumor cell surface receptors. An In-111 labeled somatostatin receptorbinding peptide, In-111-DTPA-D-Phe¹-octeotide, is in clinical use inmany countries for imaging tumors that express the somatostatin receptor(Baker, et al. Life Sci., 1991, 49, 1583-91 and Krenning, et al., Eur.J. Nucl. Med., 1993, 20, 716-31). Higher doses of thisradiopharmaceutical have been investigated for potential treatment ofthese types of cancer (Krenning, et al., Digestion, 1996, 57, 57-61).Several groups are investigating the use of Tc-99m labeled analogs ofIn-111-DTPA-D-Phe¹-octeotide for imaging and Re-186 labeled analogs fortherapy (Flanagan, et al., U.S. Pat. No. 5,556,939, Lyle, et al., U.S.Pat. No. 5,382,654, and Albert et al.,U.S. Pat. No. 5,650,134).

[0008] Angiogenesis is the process by which new blood vessels are formedfrom pre-existing capillaries or post capillary venules; it is animportant component of a variety of physiological processes includingovulation, embryonic development, wound repair, and collateral vasculargeneration in the myocardium. It is also central to a number ofpathological conditions such as tumor growth and metastasis, diabeticretinopathy, and macular degeneration. The process begins with theactivation of existing vascular endothelial cells in response to avariety of cytokines and growth factors. Tumor released cytokines orangiogenic factors stimulate vascular endothelial cells by interactingwith specific cell surface receptors for the factors. The activatedendothelial cells secrete enzymes that degrade the basement membrane ofthe vessels. The endothelial cells then proliferate and invade into thetumor tissue. The endothelial cells differentiate to form lumens, makingnew vessel offshoots of pre-existing vessels. The new blood vessels thenprovide nutrients to the tumor permitting further growth and a route formetastasis.

[0009] Under normal conditions, endothelial cell proliferation is a veryslow process, but it increases for a short period of time duringembryogenesis, ovulation and wound healing. This temporary increase incell turnover is governed by a combination of a number of growthstimulatory factors and growth suppressing factors. In pathologicalangiogenesis, this normal balance is disrupted resulting in continuedincreased endothelial cell proliferation. Some of the proangiogenicfactors that have been identified include basic fibroblast growth factor(bFGF), angiogenin, TGF-alpha, TGF-beta, and vascular endothelium growthfactor (VEGF). While interferon-alpha, interferon-beta andthrombospondin are examples of angiogenesis suppressors.

[0010] The proliferation and migration of endothelial cells in theextracellular matrix is mediated by interaction with a variety of celladhesion molecules (Folkman, J., Nature Medicine , 1995, 1, 27-31).Integrins are a diverse family of heterodimeric cell surface receptorsby which endothelial cells attach to the extracellular matrix, eachother and other cells. The integrin α_(v)β₃ is a receptor for a widevariety for a wide variety of extracellular matrix proteins with anexposed tripeptide Arg-Gly-Asp moiety and mediates cellular adhesion toits ligand: vitronectin, fibronectin, and fibrinogen, among others. Theintegrin α_(v)β₃ is minimally expressed on normal blood vessels, but issignificantly upregulated on vascular cells within a variety of humantumors. The role of the α_(v)β₃ receptors is to mediate the interactionof the endothelial cells and the extracellular matrix and facilitate themigration of the cells in the direction of the angiogenic signal, thetumor cell population. Angiogenesis induced by bFGF or TNF-alpha dependon the agency of the integrin α_(v)β₃, while angiogenesis induced byVEGF depends on the integrin α_(v)β₃ (Cheresh et. al., Science, 1955,270, 1500-2). Induction of expression of the integrins α₁β₁ and α₂β₁ onthe endothelial cell surface is another important mechanism by whichVEGF promotes angiogenesis (Senger, et. al., Proc. Natl. Acad, Sci USA,1997, 84, 13612-7).

[0011] Angiogenic factors interact with endothelial cell surfacereceptors such as the receptor tyrosine kinases EGFR, FGFR, PDGFR,Flk-1/KDR, Flt-1, Tek, tie, neuropilin-1, endoglin, endosialin, and Axl.The receptors Flk-1/KDR, neuropilin-1, and Flt-1 recognize VEGF andthese interactions play key roles in VEGF-induced angiogenesis. The Tiesubfamily of receptor tyrosine kinases are also expressed prominentlyduring blood vessel formation.

[0012] Because of the importance of angiogenesis to tumor growth andmetastasis, a number of chemotherapeutic approaches are being developedto interfere with or prevent this process. One of these approaches,involves the use of anti-angiogenic proteins such as angiostatin andendostatin. Angiostatin is a 38 kDa fragment of plasminogen that hasbeen shown in animal models to be a potent inhibitor of endothelial cellproliferation. (O'Reilly et. al., Cell, 1994, 79, 315-328) Endostatin isa 20 kDa C-terminal fragment of collagen XVIII that has also been shownto be a potent inhibitor. (O'Reilly et. al., Cell, 1997, 88, 277-285)Systemic therapy with endostatin has been shown to result in stronganti-tumor activity in animal models. However, human clinical trials ofthese two chemotherapeutic agents of biological origin have beenhampered by lack of availability.

[0013] Another approach to anti-angiogenic therapy is to use targetingmoieties that interact with endothelial cell surface receptors expressedin the angiogenic vasculature to which are attached chemotherapeuticagents. Burrows and Thorpe (Proc. Nat. Acad. Sci, USA, 1993, 90,8996-9000) described the use of an antibody-immunotoxin conjugate toeradicate tumors in a mouse model by destroying the tumor vasculature.The antibody was raised against an endothelial cell class II antigen ofthe major histocompatibility complex and was then conjugated with thecytotoxic agent, deglycosylated ricin A chain. The same group (Clin.Can. Res., 1995, 1, 1623-1634) investigated the use of antibodies raisedagainst the endothelial cell surface receptor, endoglin, conjugated todeglycosylated ricin A chain. Both of these conjugates exhibited potentanti-tumor activity in mouse models. However, both still sufferdrawbacks to routine human use. As with most antibodies or other large,foreign proteins, there is considerable risk of immunologic toxicitywhich could limit or preclude administration to humans. Also, while thevasculature targeting may improve the local concentration of theattached chemotherapeutic agents, the agents still must be cleaved fromthe antibody carrier and be transported or diffuse into the cells to becytotoxic.

[0014] Thus, it is desirable to provide anti-angiogenic pharmaceuticalsand tumor or new vasculature imaging agents which do not suffer frompoor diffusion or transportation, possible immunologic toxicity, limitedavailability, and/or a lack of specificity.

[0015] Another application of anti-angiogenic therapy is in treatingrheumatoid arthritis (RA). In RA, the ingrowth of a highly vascularizedpannus is caused by the excessive production of angiogenic factors bythe infiltrating macrophages, immune cells, or inflammatory cells.Therefore, it is desirable to have new pharmaceuticals to destroy thehighly vascularized pannus that results and thus treat the disease.

[0016] There is also a growing interest in therapeutic angiogenesis toimprove blood flow in regions of the body that have become ischemic orpoorly perfused. Several investigators are using growth factorsadministered locally to cause new vasculature to form either in thelimbs or the heart. The growth factors VEGF and bFGF are the most commonfor this application. Recent publications include: Takeshita, S., et.al., J. Clin. Invest., 1994, 93, 662-670; and Schaper, W. and Schaper,J., Collateral Circulation:Heart, Brain, Kidney, Limbs, Kluwer AcademicPublishers, Boston, 1993. The main applications that are underinvestigation in a number of laboratories are for improving cardiacblood flow and in improving peripheral vessal blood flow in the limbs.For example, Henry, T. et. al. (J. Amer. College Cardiology, 1998, 31,65A) describe the use of recombinant human VEGF in patients forimproving myocardial perfusion by therapeutic angiogenesis. Patientsreceived infusions of rhVEGF and were monitored by nuclear perfusionimaging 30 and 60 days post treatment to determine improvement inmyocardial perfusion. About 50% of patients showed improvement bynuclear perfusion imaging whereas 5/7 showed new collatoralization byangiography. Thus, it is desirable to discover a method of monitoringimproved cardiac blood flow which is targeted to new collateral vesselsthemselves and not, as in nuclear perfusion imaging, a regionalconsequence of new collateral vessels.

[0017] The detection, imaging and diagnosis of a number ofcardiovascular diseases need to be improved, including restenosis,atherosclerosis, myocardial reperfusion injury, and myocardial ischemia,stunning or infarction. It has recently been determined that in all ofthese disease conditions, the integrin receptor α_(v)β₃ plays animportant role.

[0018] For example, in the restenosis complication that occurs in˜30-50% of patients having undergone angioplasty or stent placement,neointimal hyperplasia and ultimate reocclusion is caused byaggressively proliferating vascular smooth muscle cells that expressα_(v)β₃. (Cardiovascular Res., 1997, 36, 408-428; DDT, 1997, 2, 187-199;Current Pharm. Design, 1997, 3, 545-584)

[0019] Atherosclerosis proceeds from an intial endothelial damage thatresults in the recruitment and subintimal migration of monocytes at thesite of the injury. Growth factors are released which induce medialsmooth muscle cells to proliferate and migrate to the intimal layer. Themigrating smooth muscle cells express α_(v)β₃.

[0020] In reperfusion injury, neutrophil transmigration is integrindependent and the integrins moderate initial infiltration into theviable border zone. The induction of α₅β₁, α₄β₁ and α_(v)β₅ ininfiltrating neutrophils occurs within 3 to 5 hours after reperfusion asneutrophils move from the border zone to the area of necrosis.(Circulation, 1999, 100, I-275)

[0021] Acute or chronic occlusion of a coronary artery is known toresult in angiogenesis in the heart as native collateral vessels arerecruited to attempt to relieve the ischemia. However, even a gradualocclusion usually results in areas of infarction as the resultingangiogenesis is not sufficient to prevent damage. Cardiac angiogenesishas been associated with increased expression of the growth factors VEGFand FGF and the upregulation of the growth factor receptors flt-1 andflk-1/KDR. (Drugs, 1999, 58, 391-396)

SUMMARY OF THE INVENTION

[0022] It is one object of the present invention to provide improvedanti-angiogenic pharmaceuticals, comprised of a targeting moiety thatbinds to the vitronectin receptor that is expressed in tumorneovasculature, an optional linking group, and a radioisotope. Thevitronectin receptor binding compounds target the radioisotope to thetumor neovasculature. The beta or alpha-particle emitting radioisotopeemits a cytotoxic amount of ionizing radiation which results in celldeath. The penetrating ability of radiation obviates the requirementthat the cytotoxic agent diffuse or be transported into the cell to becytotoxic.

[0023] It is another object of the present invention to providepharmaceuticals to treat rheumatoid arthritis. These pharmaceuticalscomprise a targeting moiety that binds to a receptor that is upregulatedduring angiogenesis, an optional linking group, and a radioisotope thatemits cytotoxic radiation (i.e., beta particles, alpha particles andAuger or Coster-Kronig electrons). In rheumatoid arthritis, the ingrowthof a highly vascularized pannus is caused by the excessive production ofangiogenic factors by the infiltrating macrophages, immune cells, orinflammatory cells. Therefore, the radiopharmaceuticals of the presentinvention that emit cytotoxic radiation could be used to destroy the newangiogenic vasculature that results and thus treat the disease.

[0024] It is another object of the present invention to provide imagingagents, comprised of vitronectin receptor binding compounds conjugatedto an imageable moiety, such as a gamma ray or positron emittingradioisotope, a magnetic resonance imaging contrast agent, an X-raycontrast agent, or an ultrasound contrast agent. These imaging agentsare useful for imaging tumor neovasculature, therapeutic angiogenesisinterventions in the heart, natural angiogenic processes in response toacute or chronic coronary vessel occlusion, restenosis post-angioplasty,atherosclerosis and plaque formation, and reperfusion injury.

[0025] It is another object of the present invention to providecompounds useful for preparing the pharmaceuticals of the presentinvention. These compounds are comprised of a non-peptidebenzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenecontaining targeting moiety that binds to a receptor that is upregulatedduring angiogenesis or during cardiovascular diseases, Q, an optionallinking group, L_(n), and a metal chelator or bonding moiety, C_(h). Thecompounds may have one or more protecting groups attached to the metalchelator or bonding moiety. The protecting groups provide improvedstability to the reagents for long-term storage and are removed eitherimmediately prior to or concurrent with the synthesis of theradiopharmaceuticals. Alternatively, the compounds of the presentinvention are comprised of a peptide or peptidomimetic targeting moietythat binds to a receptor that is upregulated during angiogenesis orduring cardiovascular diseases, Q, an optional linking group, L_(n), anda surfactant, S_(f).

[0026] The pharmaceuticals of the present invention may be used fordiagnostic and/or therapeutic purposes. Diagnostic radiopharmaceuticalsof the present invention are pharmaceuticals comprised of adiagnostically useful radionuclide (i.e., a radioactive metal ion thathas imageable gamma ray or positron emissions). Therapeuticradiopharmaceuticals of the present invention are pharmaceuticalscomprised of a therapeutically useful radionuclide, a radioactive metalion that emits ionizing radiation such as beta particles, alphaparticles and Auger or Coster-Kronig electrons.

[0027] The pharmaceuticals comprising a gamma ray or positron emittingradioactive metal ion are useful for imaging tumors and by gammascintigraphy or positron emission tomography. The pharmaceuticalscomprising a gamma ray or positron emitting radioactive metal ion arealso useful for imaging therapeutic angiogenesis, natural angiogenicprocesses in response to acute or chronic coronary vessel occlusion,restenosis post-angioplasty, atherosclerosis and plaque formation, andreperfusion injury by gamma scintigraphy or positron emissiontomography. The pharmaceuticals comprising a particle emittingradioactive metal ion are useful for treating cancer by delivering acytotoxic dose of radiation to the tumors. The pharmaceuticalscomprising a particle emitting radioactive metal ion are also useful fortreating rheumatoid arthritis by destroying the formation of angiogenicvasculature. The pharmaceuticals comprising a paramagnetic metal ion areuseful as magnetic resonance imaging contrast agents. Thepharmaceuticals comprising one or more X-ray absorbing or “heavy” atomsof atomic number 20 or greater are useful as X-ray contrast agents. Thepharmaceuticals comprising a microbubble of a biocompatible gas, aliquid carrier, and a surfactant microsphere, are useful as ultrasoundcontrast agents.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Thus, in a first embodiment, the present invention provides anovel compound, comprising: a targeting moiety and a chelator, whereinthe targeting moiety is bound to the chelator, is a benzodiazepine,benzodiazepinedione, or dibenzotrihydroannulene nonpeptide, and binds toa receptor that is upregulated during angiogenesis and the compound has0-1 linking groups between the targeting moiety and chelator.

[0029] In a preferred embodiment, the receptor is the integrin α_(v)β₃or α_(v)β₅ and the compound is of the formula:

(Q)_(d)-L_(n)-C_(h) or (Q)_(d)-L_(n)-(C_(h))_(d′)

[0030] wherein, Q is a compound of Formulae (Ia), (Ib) or (Ic):

[0031] wherein:

[0032] R¹ and R³ are independently selected from the group: C₁-C₆ alkyl,benzyl, phenethyl, and a bond to L_(n); provided that one of R¹ and R³is a bond to L_(n);

[0033] R² is independently selected from the group:2-benzimidazolylmethyl, 2-guanidinoethyl, 2-amino-2-pyridyl,2-amino-2-pyridylmethyl, 5-amino-2-imidazolylmethyl, and2-imidazolylmethyl;

[0034] R⁴ is independently selected from H, C₁₋₆ alkyl or benzyl;

[0035] R^(2a) is (CH₂)₃R^(3a);

[0036] R^(3a) is selected from the group:

[0037] R^(4a) is independently selected from C₁₋₆ alkyl substituted witha bond to L_(n) or benzyl substituted with a bond to L_(n);

[0038] R^(2b) is independently selected from the group:

[0039] the asterisks * denote optional positions for attaching L_(n);

[0040] or Q is a peptide selected from the group:

[0041] R^(1p) is L-valine, D-valine or L-lysine optionally substitutedon the. amino group with a bond to L_(n);

[0042] R^(2p) is L-phenylalanine, D-phenylalanine, D-1-naphthylalanine,2-aminothiazole-4-acetic acid or tyrosine, the tyrosine optionallysubstituted on the hydroxy group with a bond to L_(n);

[0043] R^(3p) is D-valine;

[0044] R^(4p) is D-tyrosine substituted on the hydroxy group with a bondto L_(n);

[0045] provided that one of R^(1p) and R^(2p) in each Q is substitutedwith a bond to L_(n), and further provided that when R^(2p) is2-aminothiazole-4-acetic acid, K is N-methylarginine;

[0046] provided that at least one Q is a compound of Formula Ia Ib, orIc;

[0047] d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0048] d′ is 1-100;

[0049] L_(n) is a linking group having the formula:

((W)_(h)—(CR⁶R⁷)_(g))_(x)-(Z)_(k)-((CR^(6a)R^(7a))_(g′)—(W)_(h′))_(x′);

[0050] W is independently selected at each occurrence from the group: O,S, NH, NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)N R⁸, C(═O), C(═O)O, OC(═O),NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′),(OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′);

[0051] aa is independently at each occurrence an amino acid;

[0052] Z is selected from the group: aryl substituted with 0-3 R¹⁰,C₃₋₁₀ cycloalkyl substituted with 0-3 R¹⁰, and a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O and substituted with 0-3 R¹⁰;

[0053] R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at eachoccurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkylsubstituted with 0-3 R¹⁰, aryl substituted with 0-3 R¹⁰, benzylsubstituted with 0-3 R¹⁰, and C₁-C₅ alkoxy substituted with 0-3 R¹⁰,NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to C_(h);

[0054] R¹⁰ is independently selected at each occurrence from the group:a bond to C_(h), COOR¹¹, C(═O)NHR¹¹, NHC(═O)R¹¹, OH, NHR¹¹, SO₃H, PO₃H,—OPO₃H₂, —OSO₃H, aryl substituted with 0-3 R¹¹, C₁₋₅ alkyl substitutedwith 0-1 R¹², C₁₋₅ alkoxy substituted with 0-1 R¹², and a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O and substituted with 0-3 R¹¹;

[0055] R¹¹ is independently selected at each occurrence from the group:H, alkyl substituted with 0-1 R¹², aryl substituted with 0-1 R¹², a 5-10membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O and substituted with 0-1 R¹²,C₃₋₁₀ cycloalkyl substituted with 0-1 R¹², polyalkylene glycolsubstituted with 0-1 R¹², carbohydrate substituted with 0-1 R¹²,cyclodextrin substituted with 0-1 R¹², amino acid substituted with 0-1R¹², polycarboxyalkyl substituted with 0-1 R¹², polyazaalkyl substitutedwith 0-1 R¹², peptide substituted with 0-1 R¹², wherein the peptide iscomprised of 2-10 amino acids, 3,6-O-disulfo-B-D-galactopyranosyl,bis(phosphonomethyl)glycine, and a bond to C_(h);

[0056] R¹² is a bond to C_(h);

[0057] k is selected from 0, 1, and 2;

[0058] h is selected from 0, 1, and 2;

[0059] h′ is selected from 0, 1, and 2;

[0060] g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0061] g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0062] s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0063] s′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0064] s″ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0065] t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0066] t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0067] x is selected from 0, 1, 2, 3, 4, and 5;

[0068] x′ is selected from 0, 1, 2, 3, 4, and 5;

[0069] C_(h) is a metal bonding unit having a formula selected from thegroup:

[0070] A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected ateach occurrence from the group: NR¹³, NR¹³R¹⁴, S, SH, S(Pg), O, OH,PR¹³, PR¹³R¹⁴, P(O)R¹⁵R¹⁶, and a bond to L_(n);

[0071] E is a bond, CH, or a spacer group independently selected at eachoccurrence from the group: C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, arylsubstituted with 0-3 R¹⁷, C₃₋₁₀ cycloalkyl substituted with 0-3 R¹⁷,heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, wherein theheterocyclo group is a 5-10 membered heterocyclic ring system containing1-4 heteroatoms independently selected from N, S, and O, C₆₋₁₀aryl-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀aryl-substituted with 0-3 R¹⁷, and a 5-10 membered heterocyclic ringsystem containing 1-4 heteroatoms independently selected from N, S, andO and substituted with 0-3 R¹⁷;

[0072] R¹³ and R¹⁴ are each independently selected from the group: abond to L_(n), hydrogen, C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, arylsubstituted with 0-3 R¹⁷, C₁₋₁₀ cycloalkyl substituted with 0-3 R¹⁷,heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, wherein theheterocyclo group is a 5-10 membered heterocyclic ring system containing1-4 heteroatoms independently selected from N, S, and O, C₆₋₁₀aryl-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀aryl-substituted with 0-3 R¹⁷, a 5-10 membered heterocyclic ring systemcontaining 1-4 heteroatoms independently selected from N, S, and O andsubstituted with 0-3 R¹⁷, and an electron, provided that when one of R¹³or R¹⁴ is an electron, then the other is also an electron;

[0073] alternatively, R¹³ and R¹⁴ combine to form ═C(R²⁰) (R²¹);

[0074] R¹⁵ and R¹⁶ are each independently selected from the group: abond to L_(n), —OH, C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, C₁-C₁₀ alkylsubstituted with 0-3 R¹⁷, aryl substituted with 0-3 R¹⁷, C₃₋₁₀cycloalkyl substituted with 0-3 R¹⁷, heterocyclo-C₁₋₁₀ alkyl substitutedwith 0-3 R¹⁷, wherein the heterocyclo group is a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀ aryl-substituted with 0-3 R¹⁷, and a 5-10membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O and substituted with 0-3 R¹⁷;

[0075] R¹⁷ is independently selected at each occurrence from the group:a bond to L_(n),═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R¹⁸, —C(═O)R¹⁸,—C(═O)N(R¹⁸)₂, —CHO, —CH₂OR¹⁸, —OC(═O)R¹⁸, —OC(═O)OR^(18a), —OR¹⁸,—OC(═O)N(R¹⁸)₂, —NR¹⁹C(═O)R¹⁸, —NR¹⁹C(═O)OR^(18a), —NR¹⁹C(═O)N(R¹⁸)₂,—NR¹⁹SO₂N(R¹⁸)₂, —NR¹⁹SO₂R^(18a), —SO₃H, —SO₂R^(18a), —SR¹⁸,—S(═O)R^(18a), —SO₂N(R¹⁸)₂, —N(R¹⁸)₂, —NHC(═S)NHR¹⁸, ═NOR¹⁸, NO₂,—C(═O)NHOR¹⁸, —C(═O)NHNR¹⁸R^(18a), —OCH₂CO₂H, 2-(1-morpholino)ethoxy,C₁-C₅ alkyl, C₂-C₄ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkylmethyl,C₂-C₆ alkoxyalkyl, aryl substituted with 0-2 R¹⁸, and a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O;

[0076] R¹⁸, R^(18a), and R¹⁹ are independently selected at eachoccurrence from the group: a bond to L_(n), H, C₁-C₆ alkyl, phenyl,benzyl, C₁-C₆ alkoxy, halide, nitro, cyano, and trifluoromethyl;

[0077] Pg is a thiol protecting group;

[0078] R²⁰ and R²¹ are independently selected from the group: H, C₁-C₁₀alkyl, —CN, —CO₂R²⁵, —C(═O)R²⁵, —C(═O)N(R²⁵)₂, C₂-C₁₀ 1-alkenesubstituted with 0-3 R²³, C₂-C₁₀ 1-alkyne substituted with 0-3 R²³, arylsubstituted with 0-3 R²³, unsaturated 5-10 membered heterocyclic ringsystem containing 1-4 heteroatoms independently selected from N, S, andO and substituted with 0-3 R²³, and unsaturated C₃₋₁₀ carbocyclesubstituted with 0-3 R²³;

[0079] alternatively, R²⁰ and R²¹, taken together with the divalentcarbon radical to which they are attached form:

[0080] R²² and R²³ are independently selected from the group: H, R²⁴,C₁-C₁₀ alkyl substituted with 0-3 R²⁴, C₂-C₁₀ alkenyl substituted with0-3 R²⁴, C₂-C₁₀ alkynyl substituted with 0-3 R²⁴, aryl substituted with0-3 R²⁴, a 5-10 membered heterocyclic ring system containing 1-4heteroatoms independently selected from N, S, and O and substituted with0-3 R²⁴, and C₃₋₁₀ carbocycle substituted with 0-3 R²⁴;

[0081] alternatively, R²², R²³ taken together form a fused aromatic or a5-10 membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O;

[0082] a and b indicate the positions of optional double bonds and n is0 or 1;

[0083] R²⁴ is independently selected at each occurrence from the group:═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R²⁵, —C(═O)R²⁵, —C(═O)N(R²⁵)₂, —N(R²⁵)₃⁺, —CH₂OR²⁵, —OC(═O)R²⁵, —OC(═O)OR^(25a), —OR²⁵, —OC(═O)N(R²⁵)₂,—NR²⁶C(═O)R²⁵, —NR²⁶C(═O)OR^(25a), —NR²⁶C(═O)N(R²⁵)₂, —NR²⁶SO₂N(R²⁵)₂,—NR²⁶SO₂R^(25a), —SO₃H, —SO₂R^(25a), —SR²⁵, —S(═O)R^(25a), —SO₂N(R²⁵)₂,—N(R²⁵)₂, ═NOR²⁵, —C(═O)NHOR²⁵, —OCH₂CO₂H, and 2-(1-morpholino)ethoxy;and,

[0084] R²⁵, R^(25a), and R²⁶ are each independently selected at eachoccurrence from the group: hydrogen and C₁-C₆ alkyl;

[0085] and a pharmaceutically acceptable salt thereof.

[0086] In a more preferred embodiment, the present invention provides acompound wherein:

[0087] d is selected from 1, 2, 3, 4, and 5;

[0088] d′ is 1-50;

[0089] W is independently selected at each occurrence from the group: O,NH, NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)NR⁸, C(═O), C(═O)O, OC(═O),NHC(═S)NH, NHC(═O)NH, SO₂, (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′),(OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′);

[0090] aa is independently at each occurrence an amino acid;

[0091] z is selected from the group: aryl substituted with 0-1 R¹⁰,C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁰, and a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O and substituted with 0-1 R¹⁰;

[0092] R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at eachoccurrence from the group: H, ═O, COOH, SO₃H, C₁-C₅ alkyl substitutedwith 0-1 R¹⁰, aryl substituted with 0-1 R¹⁰, benzyl substituted with 0-1R¹⁰, and C₁-C₅ alkoxy substituted with 0-1 R¹⁰, NHC(═O)R¹¹, C(═O)NHR¹¹,NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to C_(h);

[0093] k is 0 or 1;

[0094] s is selected from 0, 1, 2, 3, 4, and 5;

[0095] s′ is selected from 0, 1, 2, 3, 4, and 5;

[0096] s″ is selected from 0, 1, 2, 3, 4, and 5;

[0097] t is selected from 0, 1, 2, 3, 4, and 5;

[0098] A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected ateach occurrence from the group: NR¹³, NR¹³R¹⁴, S, SH, S(Pg), OH, and abond to L_(n);

[0099] E is a bond, CH, or a spacer group independently selected at eachoccurrence from the group: C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, arylsubstituted with 0-3 R¹⁷, C₃₋₁₀ cycloalkyl substituted with 0-3 R¹⁷, anda 5-10 membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O and substituted with 0-3 R¹⁷;

[0100] R¹³, and R¹⁴ are each independently selected from the group: abond to L_(n), hydrogen, C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, arylsubstituted with 0-3 R¹⁷, a 5-10 membered heterocyclic ring systemcontaining 1-4 heteroatoms independently selected from N, S, and O andsubstituted with 0-3 R¹⁷, and an electron, provided that when one of R¹³or R¹⁴ is an electron, then the other is also an electron;

[0101] alternatively, R¹³ and R¹⁴ combine to form ═C(R²⁰) (R²¹);

[0102] R¹⁷ is independently selected at each occurrence from the group:a bond to L_(n), ═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R¹⁸, —C(═O)R¹⁸,—C(═O)N(R¹⁸)₂, —CH₂OR¹⁸, —OC(═O)R¹⁸, —OC(═O)OR^(18a), —OR¹⁸,—OC(═O)N(R¹⁸)₂, —NR¹⁹C(═O)R¹⁸, —NR¹⁹C(═O)OR^(18a), —NR¹⁹C(═O)N(R¹⁸)₂,—NR¹⁹SO₂N(R¹⁸)₂, —NR¹⁹SO₂R^(18a), —SO₃H, —SO₂R^(18a), —S(═O)R^(18a),—SO₂N(R¹⁸)₂, —N(R¹⁸)₂, —NHC(═S)NHR¹⁸, ═NOR¹⁸, —C(═O)NHNR¹⁸R^(18a),—OCH₂CO₂H, and 2-(1-morpholino)ethoxy;

[0103] R¹⁸, R^(18a), and R¹⁹ are independently selected at eachoccurrence from the group: a bond to L_(n), H, and C₁-C₆ alkyl;

[0104] R²⁰ and R²¹ are independently selected from the group: H, C₁-C₅alkyl, —CO₂R²⁵, C₂-C₅ 1-alkene substituted with 0-3 R²³, C₂-C₅ 1-alkynesubstituted with 0-3 R²³, aryl substituted with 0-3 R²³, and unsaturated5-10 membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O and substituted with 0-3 R²³;

[0105] alternatively, R²⁰ and R²¹, taken together with the divalentcarbon radical to which they are attached form:

[0106] R²² and R²³ are independently selected from the group: H, andR²⁴;

[0107] alternatively, R²², R²³ taken together form a fused aromatic or a5-10 membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O;

[0108] R²⁴ is independently selected at each occurrence from the group:—CO₂R²⁵, —C(═O)N(R²⁵)₂, —CH₂OR²⁵, —OC(═O)R²⁵, —OR²⁵, —SO₃H, —N(R²⁵)₂,and —OCH₂CO₂H; and,

[0109] R²⁵ is independently selected at each occurrence from the group:H and C₁-C₃ alkyl.

[0110] In an even more preferred embodiment, the present inventionprovides a compound wherein:

[0111] R^(4a) is benzyl substituted with a bond to L_(n);

[0112] A¹ is selected from the group: OH, and a bond to L_(n);

[0113] A², A⁴, and A⁶ are each N;

[0114] A³, A⁵, and A⁸ are each OH;

[0115] A⁷ is a bond to L_(n) or NH-bond to L_(n);

[0116] E is a C₂ alkyl substituted with 0-1 R¹⁷;

[0117] R¹⁷ is ═O;

[0118] alternatively, C_(h) is

[0119] A¹ is selected from the group: OH, and a bond to L_(n);

[0120] A², A³ and A⁴ are each N;

[0121] A⁵, A⁶ and A⁸ are each OH;

[0122] A⁷ is a bond to L_(n);

[0123] E is a C₂ alkyl substituted with 0-1 R¹⁷;

[0124] R¹⁷ is ═O;

[0125] alternatively, C_(h) is

[0126] A¹ is NH₂ or N═C(R²⁰)(R²¹);

[0127] E is a bond;

[0128] A² is NHR¹³;

[0129] R¹³ is a heterocycle substituted with R¹⁷, the heterocycle beingselected from pyridine and pyrimidine;

[0130] R¹⁷ is selected from a bond to L_(n), C(═O)NHR¹⁸ and C(═O) R¹⁸;

[0131] R¹⁸ is a bond to L_(n);

[0132] R²⁴ is selected from the group: —CO₂R²⁵, —OR²⁵, —SO₃H, and—N(R²⁵)₂; and,

[0133] R²⁵ is independently selected at each occurrence from the group:hydrogen and methyl.

[0134] In another even more preferred embodiment, the present inventionprovides a compound selected from the group:

[0135](S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoyl amino)butanoic acid;

[0136](S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl) acetic acid;

[0137](S)-2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid;

[0138](S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic acid;

[0139](S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino) butanoic acid;

[0140](S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoic acid;

[0141](S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4,4-dihydroxypentyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoic acid;

[0142](S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-4-(2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)-4-carboxybutanoylamino)-4-carboxybutanoylamino)butanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)butanoic acid;

[0143](S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid;

[0144](S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)propyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino) hexanoylamino)butanoic acid;

[0145](S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid;

[0146](S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-carboxypropyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoic acid;

[0147]4-[N-(3-{(2R)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl](4S)-4-[(4S)-4-(N-{(1S)-1-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-3-carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}butanoylamino]butanoic acid;

[0148]2-(4-{3-[(6-{[(1E)-1-aza-2-(2-sulfophenyl)vinyl]amino}(3-pyridyl))carbonylamino]propyl}(2S)-7-{N-[2-(amidinoamino)ethyl]-N-methylcarbamoyl}-3-oxo-1H,2H,5H-benzo[f]1,4-diazepin-2-yl)aceticacid; and

[0149]2-[9-(N-{6-[(6-{[(1E)-1-aza-2-(2-sulfophenyl)vinyl]amino}(3-pyridyl))carbonylamino]hexyl}-N-(benzimidazol-2-ylmethyl)carbamoyl)(5S)-5,6,11-trihydro-dibenzo[b,e][7]annulen-5-yl]aceticacid;

[0150] or a pharmaceutically acceptable salt form thereof.

[0151] In a further preferred embodiment, the present invention providesa kit comprising a compound of the present invention, or apharmaceutically acceptable salt form thereof and a pharmaceuticallyacceptable carrier.

[0152] In an even further preferred embodiment, the kit furthercomprises one or more ancillary ligands and a reducing agent.

[0153] In a still further preferred embodiment, the ancillary ligandsare tricine and TPPTS.

[0154] In another still further preferred embodiment, the reducing agentis tin(II).

[0155] In a second embodiment, the present invention provides a noveldiagnostic or therapeutic metallopharmaceutical composition, comprising:a metal, a chelator capable of chelating the metal and a targetingmoiety, wherein the targeting moiety is bound to the chelator, is abenzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator.

[0156] In a preferred embodiment, the metallopharmaceutical is adiagnostic radiopharmaceutical, the metal is a radioisotope selectedfrom the group: ^(99m)Tc, ⁹⁵Tc, ¹¹¹Tn, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, and ⁶⁸Ga, andthe linking group is present between the targeting moiety and chelator.

[0157] In another preferred embodiment, the targeting moiety is abenzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene and thereceptor is ._(v).₃ or ._(v).₅.

[0158] In another preferred embodiment, the radioisotope is ^(99m)Tc or⁹⁵Tc, the radiopharmaceutical further comprises a first ancillary ligandand a second ancillary ligand capable of stabilizing theradiopharmaceutical.

[0159] In another preferred embodiment, the radioisotope is ^(99m)Tc.

[0160] In another preferred embodiment, the radiopharmaceutical isselected from the group:

[0161]^(99m)Tc((S)-2-(2,5-diaza-5-(6((6-(diazenido)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl) acetic acid)(tricine)(TPPTS) and

[0162]^(99m)Tc((S)-2-(2,5-diaza-9-(N-(6-((6-(diazenido)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid)(tricine)(TPPTS);

[0163] In another preferred embodiment, the radioisotope is ¹¹¹In.

[0164] In another preferred embodiment, the radiopharmaceutical isselected from the group:

[0165]¹¹¹In complex of6-(N-(3-(3-aza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-((2-((carboxymethyl)(2-((carboxymethyl)methylamino)ethyl)amino)ethyl)(2-((carboxymethyl)ethylamino)ethyl)amino)-acetylamino)-4-oxooctane-1,8-dicarboxylicacid;

[0166]¹¹¹In complex of(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino) butanoic acid; and

[0167]¹¹¹In complex of(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoic acid.

[0168] In another preferred embodiment wherein the metallopharmaceuticalis a therapeutic radiopharmaceutical, the metal is a radioisotopeselected from the group: ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y,²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy,¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir, and the linking group is presentbetween the targeting moiety and chelator.

[0169] In another preferred embodiment, the targeting moiety is abenzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene and thereceptor is α_(v)β₃ or α_(v)β₅.

[0170] In another preferred embodiment, the radioisotope is ¹⁴⁹Pm.

[0171] In another preferred embodiment, the radiopharmaceutical isselected from the group:

[0172] the Pm-149 complex of(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoyl amino)butanoic acid; and

[0173] the Pm-149 complex of(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino) butanoic acid.

[0174] In another preferred embodiment, the radioisotope is ¹⁷⁷Lu.

[0175] In another preferred embodiment, the radiopharmaceutical isselected from the group:

[0176] the Lu-177 complex of(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoyl amino)butanoic acid; and

[0177] the Lu-177 complex of(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino) butanoic acid; and

[0178] the Lu-177 complex of(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoic acid.

[0179] [24] In another preferred embodiment, the radioisotope is ⁹⁰Y

[0180] [25] In another preferred embodiment, the radiopharmaceutical isselected from the group:

[0181] the Y-90 complex of(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoyl amino)butanoic acid; and

[0182] the Y-90 complex of(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(14,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino) butanoic acid; and

[0183] the Y-90 complexof(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoic acid.

[0184] In another preferred embodiment wherein the metallopharmaceuticalis a MRI contrast agent, the metal is a paramagnetic metal ion selectedfrom the group: Gd(III), Dy(III), Fe(III), and Mn(II), and the linkinggroup is present between the targeting moiety and chelator.

[0185] In another preferred embodiment, the targeting moiety is abenzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene and thereceptor is α_(v)β₃ or α_(v)β₅.

[0186] In another preferred embodiment, the metal ion is Gd(III).

[0187] In yet another preferred embodiment wherein themetallopharmaceutical is a X-ray contrast agent, the metal is selectedfrom the group: Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au, Yb, Dy,Cu, Rh, Ag, and Ir, and the linking group is present between thetargeting moiety and chelator.

[0188] In another preferred embodiment, the present invention provides anovel method of treating rheumatoid arthritis in a patient comprising:administering a therapeutic radiopharmaceutical of claim 18 capable oflocalizing in new angiogenic vasculature to a patient by injection orinfusion.

[0189] In another preferred embodiment, the present invention provides anovel method of treating cancer in a patient comprising: administeringto a patient in need thereof a therapeutic radiopharmaceutical of claim18 by injection or infusion.

[0190] In another preferred embodiment, the present invention provides anovel method of treating restenosis in a patient comprising:administering to a patient, either systemically or locally, atherapeutic radiopharmaceutical of claim 18 capable of localizing in therestenotic area and delivering an effective dose of radiation.

[0191] In another preferred embodiment, the present invention provides anovel method of imaging cancer in a patient comprising: (1)administering a diagnostic radiopharmaceutical of claim 11 to a patientby injection or infusion; (2) imaging the patient using planar or SPECTgamma scintigraphy, or positron emission tomography.

[0192] In another preferred embodiment, the present invention provides anovel method of imaging cancer in a patient comprising: (1)administering a MRI contrast agent of claim 26; and (2) imaging thepatient using magnetic resonance imaging.

[0193] In another preferred embodiment, the present invention provides anovel method of imaging cancer in a patient comprising: (1)administering an X-ray contrast agent of claim 29; and (2) imaging thepatient using X-ray computed tomography.

[0194] In another preferred embodiment, the present invention provides anovel method of imaging therapeutic angiogenesis in a patientcomprising: (1) administering a diagnostic radiopharmaceutical, a MRIcontrast agent, or a X-ray contrast agent of claim 10 to a patient byinjection or infusion; (2) imaging the area of the patient wherein thedesired formation of new blood vessels is located.

[0195] In another preferred embodiment, the present invention provides anovel method of imaging atherosclerosis in a patient comprising: (1)administering a diagnostic radiopharmaceutical, a MRI contrast agent, ora X-ray contrast agent of claim 10 to a patient by injection orinfusion; (2) imaging the area of the patient wherein theatherosclerosis is located.

[0196] In another even more preferred embodiment, the present inventionprovides a novel method of imaging restenosis in a patient comprising:(1) administering a diagnostic radiopharmaceutical, a MRI contrastagent, or a X-ray contrast agent of claim 10 to a patient by injectionor infusion; (2) imaging the area of the patient wherein the restenosisis located.

[0197] In another even more preferred embodiment, the present inventionprovides a novel method of imaging cardiac ischemia in a patientcomprising: (1) administering a diagnostic radiopharmaceutical, a MRIcontrast agent, or a X-ray contrast agent of claim 10 to a patient byinjection or infusion; (2) imaging the area of the myocardium whereinthe ischemic region is located.

[0198] In another even more preferred embodiment, the present inventionprovides a novel method of imaging myocardial reperfusion injury in apatient comprising: (1) administering a diagnostic radiopharmaceutical,a MRI contrast agent, or a X-ray contrast agent of claim 10 to a patientby injection or infusion; (2) imaging the area of myocardium wherein thereperfusion injury is located.

[0199] In a third embodiment, the present invention provides a novelcompound, comprising: a targeting moiety and a surfactant, wherein thetargeting moiety is bound to the surfactant, is a benzodiazepine,benzodiazepinedione, or dibenzotrihydroannulene nonpeptide, and binds toa receptor that is upregulated during angiogenesis and the compound has0-1 linking groups between the targeting moiety and surfactant.

[0200] In a preferred embodiment, the receptor is the integrin α_(v)β₃or α_(v)β₅ and the compound is of the formula:

(Q)_(d)-L_(n)-S_(f)

[0201] wherein, Q is a compound of Formulae (Ia), (Ib) or (Ic):

[0202] wherein:

[0203] R¹ and R³ are independently selected from the group: C₁-C₆ alkyl,benzyl, phenethyl, and a bond to L_(n); provided that one of R¹ and R³is a bond to L_(n);

[0204] R² is independently selected from the group:2-benzimidazolylmethyl, 2-guanidinoethyl, 2-amino-2-pyridyl,2-amino-2-pyridylmethyl, 5-amino-2-imidazolylmethyl, and2-imidazolylmethyl;

[0205] R⁴ is independently selected from H, C₁₋₆ alkyl or benzyl;

[0206] R^(2a) is (CH₂)₃R^(3a);

[0207] R^(3a) is selected from the group:

[0208] R^(4a) is independently selected from C₁₋₆ alkyl substituted witha bond to L_(n) or benzyl substituted with a bond to L_(n);

[0209] R^(2b) is independently selected from the group:

[0210] the asterisks * denote optional positions for attaching L_(n);

[0211] or Q is a peptide selected from the group:

[0212] R^(1p) is L-valine, D-valine or L-lysine optionally substitutedon the .amino group with a bond to L_(n);

[0213] R^(2p) is L-phenylalanine, D-phenylalanine, D-1-naphthylalanine,2-aminothiazole-4-acetic acid or tyrosine, the tyrosine optionallysubstituted on the hydroxy group with a bond to L_(n);

[0214] R^(3p) is D-valine;

[0215] R^(4p) is D-tyrosine substituted on the hydroxy group with a bondto L_(n);

[0216] provided that one of R^(1p) and R^(2p) in each Q is substitutedwith a bond to L_(n), and further provided that when R^(2p) is2-aminothiazole-4-acetic acid, K is N-methylarginine;

[0217] provided that at least one Q is a compound of Formula Ia Ib, orIc;

[0218] d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0219] L_(n) is a linking group having the formula:

((W)_(h)-(CR⁶R⁷)_(g))_(x)-(Z)_(k)-((CR^(6a)R^(7a))_(g′)-(W)_(h′))_(x′);

[0220] W is independently selected at each occurrence from the group: O,S, NH, NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)N R⁸, C(═O), C(═O)O, OC(═O),NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, (OCH₂CH₂)₂₀₋₂₀₀, (CH₂CH₂O)₂₀₋₂₀₀,(OCH₂CH₂CH₂)₂₀₋₂₀₀, (CH₂CH₂CH₂O)₂₀₋₂₀₀, and (aa)_(t′);

[0221] aa is independently at each occurrence an amino acid;

[0222] Z is selected from the group: aryl substituted with 0-3 R¹⁰,C₃₋₁₀ cycloalkyl substituted with 0-3 R¹⁰, and a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O and substituted with 0-3 R¹⁰;

[0223] R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at eachoccurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkylsubstituted with 0-3 R¹⁰, aryl substituted with 0-3 R¹⁰, benzylsubstituted with 0-3 R¹⁰, and C₁-C₅ alkoxy substituted with 0-3 R¹⁰,NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to S_(f);

[0224] R¹⁰ is independently selected at each occurrence from the group:a bond to S_(f), COOR¹¹, C(═O)NHR¹¹, NHC(═O)R¹¹, OH, NHR¹¹, SO₃H, PO₃H,—OPO₃H₂, —OSO₃H, aryl substituted with 0-3 R¹¹, C₁₋₅ alkyl substitutedwith 0-1 R¹², C₁₋₅ alkoxy substituted with 0-1 R¹², and a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O and substituted with 0-3 R¹¹;

[0225] R¹¹ is independently selected at each occurrence from the group:H, alkyl substituted with 0-1 R¹², aryl substituted with 0-1 R¹², a 5-10membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O and substituted with 0-1 R¹²,C₃₋₁₀ cycloalkyl substituted with 0-1 R¹², and a bond to S_(f);

[0226] R¹² is a bond to S_(f);

[0227] k is selected from 0, 1, and 2;

[0228] h is selected from 0, 1, and 2;

[0229] h′ is selected from 0, 1, and 2;

[0230] g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0231] g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0232] t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0233] x is selected from 0, 1, 2, 3, 4, and 5;

[0234] x′ is selected from 0, 1, 2, 3, 4, and 5;

[0235] S_(f) is a surfactant which is a lipid or a compound of theformula:

[0236] A⁹ is selected from the group: OH and OR²⁷;

[0237] A¹⁰ is OR²⁷;

[0238] R²⁷ is C(═O)C₁₋₂₀ alkyl;

[0239] E¹ is C₁₋₁₀ alkylene substituted with 1-3 R²⁸;

[0240] R²⁸ is independently selected at each occurrence from the group:R³⁰, —PO₃H—R³⁰, ═O, —CO₂R²⁹, —C(═O)R²⁹, —C(═O)N(R²⁹)₂, —CH₂OR²⁹, —OR²⁹,—N(R²⁹)₂, C₁-C₅ alkyl, and C₂-C₄ alkenyl;

[0241] R²⁹ is independently selected at each occurrence from the group:R³⁰, H, C₁-C₆ alkyl, phenyl, benzyl, and trifluoromethyl;

[0242] R³⁰ is a bond to L_(n);

[0243] and a pharmaceutically acceptable salt thereof.

[0244] In another preferred embodiment, the compound is of the formula:

Q-L_(n)-S_(f)

[0245] wherein: Q is a compound of Formulae (Ia), (Ib), or (Ic):

[0246] R^(4a) is benzyl substituted with a bond to L_(n);

[0247] R^(2b) is

[0248] Z is selected from the group: aryl substituted with 0-1 R¹⁰,C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁰, and a 5-10 memberedheterocyclic ring system containing 1-4 heteroatoms independentlyselected from N, S, and O and substituted with 0-1 R¹⁰;

[0249] R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at eachoccurrence from the group: H, ═O, COOH, SO₃H, C₁-C₅ alkyl substitutedwith 0-1 R¹⁰, aryl substituted with 0-1 R¹⁰, benzyl substituted with 0-1R¹⁰, and C₁-C₅ alkoxy substituted with 0-1 R¹⁰, NHC(═O)R¹¹, C(═O)NHR¹¹,NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to S_(f);

[0250] k is 0 or 1;

[0251] S_(f) is a surfactant which is a lipid or a compound of theformula:

[0252] A⁹ is OR²⁷;

[0253] A¹⁰ is OR²⁷;

[0254] R²⁷ is C(═O)C₁₋₁₅ alkyl;

[0255] E¹ is C₁₋₄ alkylene substituted with 1-3 R²⁸;

[0256] R²⁸ is independently selected at each occurrence from the group:R³⁰, —PO₃H—R³⁰, ═O, —CO₂R²⁹, —C(═O)R²⁹, —CH₂OR²⁹, —OR²⁹, and C₁-C₅alkyl;

[0257] R²⁹ is independently selected at each occurrence from the group:R³⁰, H, C₁-C₆ alkyl, phenyl, and benzyl;

[0258] R³⁰ is a bond to L_(n);

[0259] and a pharmaceutically acceptable salt thereof.

[0260] In another preferred embodiment, the compound selected from thegroup:

[0261] Sodium1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid-dodecoanoate conjugate;

[0262] DPPE-PEG₃₄₀₀-[(S) -2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid]-dodecoanoate conjugate; and

[0263][(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)carbamoyl)propyl)carbamoyl]-w-amino-PEG₃₄₀₀-dodecanoate-DPPEconjugate.

[0264] In another more preferred embodiment, the present inventionprovides a novel ultrasound contrast agent composition, comprising:

[0265] (a) a compound of claim 41, comprising: a benzodiazepine,benzodiazepinedione, or dibenzotrihydroannulene that binds to theintegrin α_(v)β₃, or α_(v)β₅, a surfactant and a linking group betweenthe benzodiazepine and the surfactant;

[0266] (b) a parenterally acceptable carrier; and,

[0267] (c) an echogenic gas.

[0268] In another preferred embodiment, the present invention provides anovel ultrasound contrast agent composition, further comprising:1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, andN-(methoxypolyethylene glycol 5000carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.

[0269] In another preferred embodiment, the echogenic gas is a C₂₋₅perfluorocarbon.

[0270] In another preferred embodiment, the present invention provides amethod of imaging cancer in a patient comprising: (1) administering, byinjection or infusion, a ultrasound contrast agent composition of claim45 to a patient; and (2) imaging the patient using sonography.

[0271] In another preferred embodiment, the present invention provides amethod of imaging therapeutic angiogenesis in a patient comprising: (1)administering, by injection or infusion, an ultrasound contrast agentcomposition of claim 45 to a patient; (2) imaging the area of thepatient wherein the desired formation of new blood vessels is located.

[0272] In another preferred embodiment, the present invention provides amethod of imaging atherosclerosis in a patient comprising: (1)administering, by injection or infusion, an ultrasound contrast agentcomposition of claim 45 to a patient; (2) imaging the area of thepatient wherein the atherosclerosis is located.

[0273] In another preferred embodiment, the present invention provides amethod of imaging restenosis in a patient comprising: (1) administering,by injection or infusion, an ultrasound contrast agent composition ofclaim 45 to a patient; (2) imaging the area of the patient wherein therestenosis is located.

[0274] [52] In another preferred embodiment, the present inventionprovides a method of imaging cardiac ischemia in a patient comprising:(1) administering, by injection or infusion, an ultrasound contrastagent composition of claim 45 to a patient; (2) imaging the area of themyocardium wherein the ischemic region is located.

[0275] In another preferred embodiment, the present invention provides amethod of imaging myocardial reperfusion injury in a patient comprising:(1) administering, by injection or infusion, an ultrasound contrastagent composition of claim 45 to a patient; (2) imaging the area ofmyocardium wherein the reperfusion injury is located.

[0276] In another preferred embodiment, the present invention provides anovel therapeutic radiopharmaceutical composition, comprising:

[0277] (a) a therapeutic radiopharmaceutical of claim 19; and,

[0278] (b) a parenterally acceptable carrier.

[0279] In another preferred embodiment, the present invention provides anovel diagnostic radiopharmaceutical composition, comprising:

[0280] (a) a diagnostic radiopharmaceutical, a MRI contrast agent, or aX-ray contrast agent of claim 10; and,

[0281] (b) a parenterally acceptable carrier.

[0282] Another aspect of the present invention are diagnostic kits forthe preparation of radiopharmaceuticals useful as imaging agents forcancer. Diagnostic kits of the present invention comprise one or morevials containing the sterile, non-pyrogenic, formulation comprised of apredetermined amount of a reagent of the present invention, andoptionally other components such as one or two ancillary ligands,reducing agents, transfer ligands, buffers, lyophilization aids,stabilization aids, solubilization aids and bacteriostats. The inclusionof one or more optional components in the formulation will frequentlyimprove the ease of synthesis of the radiopharmaceutical by thepracticing end user, the ease of manufacturing the kit, the shelf-lifeof the kit, or the stability and shelf-life of the radiopharmaceutical.The inclusion of one or two ancillary ligands is required for diagnostickits comprising reagent comprising a hydrazine or hydrazone bondingmoiety. The one or more vials that contain all or part of theformulation can independently be in the form of a sterile solution or alyophilized solid.

[0283] Another aspect of the present invention contemplates a method ofimaging cancer in a patient involving: (1) synthesizing a diagnosticradiopharmaceutical of the present invention, using a reagent of thepresent invention, capable of localizing in tumors; (2) administeringsaid radiopharmaceutical to a patient by injection or infusion; (3)imaging the patient using planar or SPECT gamma scintigraphy, orpositron emission tomography.

[0284] Another aspect of the present invention contemplates a method ofimaging cancer in a patient involving: (1) administering a paramagneticmetallopharmaceutical of the present invention capable of localizing intumors to a patient by injection or infusion; and (2) imaging thepatient using magnetic resonance imaging.

[0285] Another aspect of the present invention contemplates a method ofimaging cancer in a patient involving: (1) administering a X-raycontrast agent of the present invention capable of localizing in tumorsto a patient by injection or infusion; and (2) imaging the patient usingX-ray computed tomography.

[0286] Another aspect of the present invention contemplates a method ofimaging cancer in a patient involving: (1) administering a ultrasoundcontrast agent of the present invention capable of localizing in tumorsto a patient by injection or infusion; and (2) imaging the patient usingsonography.

[0287] Another aspect of the present invention contemplates a method oftreating cancer in a patient involving: (1) administering a therapeuticradiopharmaceutical of the present invention capable of localizing intumors to a patient by injection or infusion.

Definitions

[0288] The compounds herein described may have asymmetric centers.Unless otherwise indicated, all chiral, diastereomeric and racemic formsare included in the present invention. Many geometric isomers ofolefins, C═N double bonds, and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present invention. It will be appreciated that compounds of thepresent invention contain asymmetrically substituted carbon atoms, andmay be isolated in optically active or racemic forms. It is well knownin the art how to prepare optically active forms, such as by resolutionof racemic forms or by synthesis from optically active startingmaterials. Two distinct isomers (cis and trans) of the peptide bond areknown to occur; both can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentinvention. The D and L-isomers of a particular amino acid are designatedherein using the conventional 3-letter abbreviation of the amino acid,as indicated by the following examples: D-Leu, or L-Leu.

[0289] When any variable occurs more than one time in any substituent orin any formula, its definition on each occurrence is independent of itsdefinition at every other occurrence. Thus, for example, if a group isshown to be substituted with 0-2 R⁵², then said group may optionally besubstituted with up to two R⁵², and R⁵² at each occurrence is selectedindependently from the defined list of possible R⁵². Also, by way ofexample, for the group —N(R⁵³)₂, each of the two R⁵³ substituents on Nis independently selected from the defined list of possible R⁵³.Combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds. When a bond to asubstituent is shown to cross the bond connecting two atoms in a ring,then such substituent may be bonded to any atom on the ring.

[0290] The term “nonpeptide” means preferably less than three amidebonds in the backbone core of the targeting moiety or preferably lessthan three amino acids or amino acid mimetics in the targeting moiety.

[0291] The term “metallopharmaceutical” means a pharmaceuticalcomprising a metal. The metal is the cause of the imageable signal indiagnostic applications and the source of the cytotoxic radiation inradiotherapeutic applications. Radiopharmaceuticals aremetallopharmaceuticals in which the metal is a radioisotope.

[0292] By “reagent” is meant a compound of this invention capable ofdirect transformation into a metallopharmaceutical of this invention.Reagents may be utilized directly for the preparation of themetallopharmaceuticals of this invention or may be a component in a kitof this invention.

[0293] The term “binding agent” means a metallopharmaceutical of thisinvention having affinity for and capable of binding to the vitronectinreceptor. The binding agents of this invention have Ki<1000 nM.

[0294] By “stable compound” or “stable structure” is meant herein acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious pharmaceutical agent.

[0295] The term “substituted”, as used herein, means that one or morehydrogens on the designated atom or group is replaced with a selectionfrom the indicated group, provided that the designated atom's or group'snormal valency is not exceeded, and that the substitution results in astable compound. When a substituent is keto (i.e., ═O), then 2 hydrogenson the atom are replaced.

[0296] The term “bond”, as used herein, means either a single or doublebond.

[0297] The term “salt”, as used herein, is used as defined in the CRCHandbook of Chemistry and Physics, 65th Edition, CRC Press, Boca Raton,Fla., 1984, as any substance which yields ions, other than hydrogen orhydroxyl ions. As used herein, “pharmaceutically acceptable salts” referto derivatives of the disclosed compounds modified by making acid orbase salts. Examples of pharmaceutically acceptable salts include, butare not limited to, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of acidic residues such as carboxylicacids; and the like.

[0298] The phrase “pharmaceutically acceptable” is employed herein torefer to those compounds, materials, compositions, and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

[0299] As used herein, “pharmaceutically acceptable salts” refer toderivatives of the disclosed compounds wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, nitric and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like.

[0300] The pharmaceutically acceptable salts of the present inventioncan be synthesized from the parent compound which contains a basic oracidic moiety by conventional chemical methods. Generally, such saltscan be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17th ed., Mack PublishingCompany, Easton, Pa., 1985, p. 1418, the disclosure of which is herebyincorporated by reference.

[0301] As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, examples of which include, but are notlimited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl;“cycloalkyl” or “carbocycle” is intended to include saturated andpartially unsaturated ring groups, including mono-, bi- or poly-cyclicring systems, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl and adamantyl; “bicycloalkyl” or “bicyclic” isintended to include saturated bicyclic ring groups such as[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane(decalin), [2.2.2]bicyclooctane, and so forth.

[0302] As used herein, the term “alkene” or “alkenyl” is intended toinclude hydrocarbon chains having the specified number of carbon atomsof either a straight or branched configuration and one or moreunsaturated carbon-carbon bonds which may occur in any stable pointalong the chain, such as ethenyl, propenyl, and the like.

[0303] As used herein, the term “alkyne” or “alkynyl” is intended toinclude hydrocarbon chains having the specified number of carbon atomsof either a straight or branched configuration and one or moreunsaturated carbon-carbon triple bonds which may occur in any stablepoint along the chain, such as propargyl, and the like.

[0304] As used herein, “aryl” or “aromatic residue” is intended to meanphenyl or naphthyl, which when substituted, the substitution can be atany position.

[0305] As used herein, the term “heterocycle” or “heterocyclic system”is intended to mean a stable 5- to 7-membered monocyclic or bicyclic or7- to 10-membered bicyclic heterocyclic ring which is saturatedpartially unsaturated or unsaturated (aromatic), and which consists ofcarbon atoms and from 1 to 4 heteroatoms independently selected from thegroup consisting of N, O and S and including any bicyclic group in whichany of the above-defined heterocyclic rings is fused to a benzene ring.The nitrogen and sulfur heteroatoms may optionally be oxidized. Theheterocyclic ring may be attached to its pendant group at any heteroatomor carbon atom which results in a stable structure. The heterocyclicrings described herein may be substituted on carbon or on a nitrogenatom if the resulting compound is stable. If specifically noted, anitrogen in the heterocycle may optionally be quaternized. It ispreferred that when the total number of S and O atoms in the heterocycleexceeds 1, then these heteroatoms are not adjacent to one another. It ispreferred that the total number of S and O atoms in the heterocycle isnot more than 1. As used herein, the term “aromatic heterocyclic system”is intended to mean a stable 5- to 7- membered monocyclic or bicyclic or7- to 10-membered bicyclic heterocyclic aromatic ring which consists ofcarbon atoms and from 1 to 4 heteroatoms independently selected from thegroup consisting of N, O and S. It is preferred that the total number ofS and O atoms in the aromatic heterocycle is not more than 1.

[0306] Examples of heterocycles include, but are not limited to,1H-indazole, 2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl,3H-indolyl, 4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl,6H-1,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,benzisothiazolyl, benzimidazalonyl, carbazolyl, 4aH-carbazolyl,β-carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl,furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl.,oxazolyl, oxazolidinylperimidinyl, phenanthridinyl, phenanthrolinyl,phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl,phthalazinyl, piperazinyl, piperidinyl, pteridinyl, piperidonyl,4-piperidonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole,pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl,pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl,quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl,tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl,xanthenyl. Preferred heterocycles include, but are not limited to,pyridinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, indolyl,benzimidazolyl, 1H-indazolyl, oxazolidinyl, benzotriazolyl,benzisoxazolyl, oxindolyl, benzoxazolinyl, or isatinoyl. Also includedare fused ring and spiro compounds containing, for example, the aboveheterocycles.

[0307] As used herein, the term “alkaryl” means an aryl group bearing analkyl group of 1-10 carbon atoms; the term “aralkyl” means an alkylgroup of 1-10 carbon atoms bearing an aryl group; the term “arylalkaryl”means an aryl group bearing an alkyl group of 1-10 carbon atoms bearingan aryl group; and the term “heterocycloalkyl” means an alkyl group of1-10 carbon atoms bearing a heterocycle.

[0308] A “polyalkylene glycol” is a polyethylene glycol, polypropyleneglycol or polybutylene glycol having a molecular weight of less thanabout 5000, terminating in either a hydroxy or alkyl ether moiety.

[0309] A “carbohydrate” is a polyhydroxy aldehyde, ketone, alcohol oracid, or derivatives thereof, including polymers thereof havingpolymeric linkages of the acetal type.

[0310] A “cyclodextrin” is a cyclic oligosaccharide. Examples ofcyclodextrins include, but are not limited to, α-cyclodextrin,hydroxyethyl-α-cyclodextrin, hydroxypropyl-α-cyclodextrin,β-cyclodextrin, hydroxypropyl-β-cyclodextrin,carboxymethyl-β-cyclodextrin, dihydroxypropyl-β-cyclodextrin,hydroxyethyl-β-cyclodextrin, 2,6 di-O-methyl-β-cyclodextrin,sulfated-β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin,dihydroxypropyl-γ-cyclodextrin, hydroxyethyl-γ-cyclodextrin, andsulfated γ-cyclodextrin.

[0311] As used herein, the term “polycarboxyalkyl” means an alkyl grouphaving between two and about 100 carbon atoms and a plurality ofcarboxyl substituents; and the term “polyazaalkyl” means a linear orbranched alkyl group having between two and about 100 carbon atoms,interrupted by or substituted with a plurality of amine groups.

[0312] A “reducing agent” is a compound that reacts with a radionuclide,which is typically obtained as a relatively unreactive, high oxidationstate compound, to lower its oxidation state by transferring electron(s)to the radionuclide, thereby making it more reactive. Reducing agentsuseful in the preparation of radiopharmaceuticals and in diagnostic kitsuseful for the preparation of said radiopharmaceuticals include but arenot limited to stannous chloride, stannous fluoride, formamidinesulfinic acid, ascorbic acid, cysteine, phosphines, and cuprous orferrous salts. Other reducing agents are described in Brodack et. al.,PCT Application 94/22496, which is incorporated herein by reference.

[0313] A “transfer ligand” is a ligand that forms an intermediatecomplex with a metal ion that is stable enough to prevent unwantedside-reactions but labile enough to be converted to ametallopharmaceutical. The formation of the intermediate complex iskinetically favored while the formation of the metallopharmaceutical isthermodynamically favored. Transfer ligands useful in the preparation ofmetallopharmaceuticals and in diagnostic kits useful for the preparationof diagnostic radiopharmaceuticals include but are not limited togluconate, glucoheptonate, mannitol, glucarate,N,N,N′,N′-ethylenediaminetetraacetic acid, pyrophosphate andmethylenediphosphonate. In general, transfer ligands are comprised ofoxygen or nitrogen donor atoms.

[0314] The term “donor atom” refers to the atom directly attached to ametal by a chemical bond.

[0315] “Ancillary” or “co-ligands” are ligands that are incorporatedinto a radiopharmaceutical during its synthesis. They serve to completethe coordination sphere of the radionuclide together with the chelatoror radionuclide bonding unit of the reagent. For radiopharmaceuticalscomprised of a binary ligand system, the radionuclide coordinationsphere is composed of one or more chelators or bonding units from one ormore reagents and one or more ancillary or co-ligands, provided thatthere are a total of two types of ligands, chelators or bonding units.For example, a radiopharmaceutical comprised of one chelator or bondingunit from one reagent and two of the same ancillary or co-ligands and aradiopharmaceutical comprised of two chelators or bonding units from oneor two reagents and one ancillary or co-ligand are both considered to becomprised of binary ligand systems. For radiopharmaceuticals comprisedof a ternary ligand system, the radionuclide coordination sphere iscomposed of one or more chelators or bonding units from one or morereagents and one or more of two different types of ancillary orco-ligands, provided that there are a total of three types of ligands,chelators or bonding units. For example, a radiopharmaceutical comprisedof one chelator or bonding unit from one reagent and two differentancillary or co-ligands is considered to be comprised of a ternaryligand system.

[0316] Ancillary or co-ligands useful in the preparation ofradiopharmaceuticals and in diagnostic kits useful for the preparationof said radiopharmaceuticals are comprised of one or more oxygen,nitrogen, carbon, sulfur, phosphorus, arsenic, selenium, and telluriumdonor atoms. A ligand can be a transfer ligand in the synthesis of aradiopharmaceutical and also serve as an ancillary or co-ligand inanother radiopharmaceutical. Whether a ligand is termed a transfer orancillary or co-ligand depends on whether the ligand remains in theradionuclide coordination sphere in the radiopharmaceutical, which isdetermined by the coordination chemistry of the radionuclide and thechelator or bonding unit of the reagent or reagents.

[0317] A “chelator” or “bonding unit” is the moiety or group on areagent that binds to a metal ion through the formation of chemicalbonds with one or more donor atoms.

[0318] The term “binding site” means the site in vivo or in vitro thatbinds a biologically active molecule.

[0319] A “diagnostic kit” or “kit” comprises a collection of components,termed the formulation, in one or more vials which are used by thepracticing end user in a clinical or pharmacy setting to synthesizediagnostic radiopharmaceuticals. The kit provides all the requisitecomponents to synthesize and use the diagnostic radiopharmaceuticalexcept those that are commonly available to the practicing end user,such as water or saline for injection, a solution of the radionuclide,equipment for heating the kit during the synthesis of theradiopharmaceutical, if required, equipment necessary for administeringthe radiopharmaceutical to the patient such as syringes and shielding,and imaging equipment.

[0320] Therapeutic radiopharmaceuticals, X-ray contrast agentpharmaceuticals, ultrasound contrast agent pharmaceuticals andmetallopharmaceuticals for magnetic resonance imaging contrast areprovided to the end user in their final form in a formulation containedtypically in one vial, as either a lyophilized solid or an aqueoussolution. The end user reconstitutes the lyophilized with water orsaline and withdraws the patient dose or just withdraws the dose fromthe aqueous solution formulation as provided.

[0321] A “lyophilization aid” is a component that has favorable physicalproperties for lyophilization, such as the glass transition temperature,and is added to the formulation to improve the physical properties ofthe combination of all the components of the formulation forlyophilization.

[0322] A “stabilization aid” is a component that is added to themetallopharmaceutical or to the diagnostic kit either to stabilize themetallopharmaceutical or to prolong the shelf-life of the kit before itmust be used. Stabilization aids can be antioxidants, reducing agents orradical scavengers and can provide improved stability by reactingpreferentially with species that degrade other components or themetallopharmaceutical.

[0323] A “solubilization aid” is a component that improves thesolubility of one or more other components in the medium required forthe formulation.

[0324] A “bacteriostat” is a component that inhibits the growth ofbacteria in a formulation either during its storage before use of aftera diagnostic kit is used to synthesize a radiopharmaceutical.

[0325] The following abbreviations are used herein: Acm acetamidomethylb-Ala, beta-Ala 3-aminopropionic acid  or bAla ATA2-aminothiazole-5-acetic acid or 2-  aminothiazole-5-acetyl group Boct-butyloxycarbonyl CBZ, Cbz or Z Carbobenzyloxy Cit citrulline Dap2,3-diaminopropionic acid DCC dicyclohexylcarbodiimide DIEAdiisopropylethylamine DMAP 4-dimethylaminopyridine EOE ethoxyethyl HBTU2-(1H-Benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphatehynic boc-hydrazinonicotinyl group or 2- [[[5-  [carbonyl]-2-pyridinyl]hydrazono]methyl]-  benzenesulfonic acid, NMeArg or MeArga-N-methyl arginine NMeAsp a-N-methyl aspartic acid NNMN-methylmorpholine OcHex O-cyclohexyl OBzl O-benzyl oSu O-succinimidylTBTU 2-(1H-Benzotriazol-1-yl)1,1,3,3- tetramethyluroniumtetrafluoroborate THF tetrahydrofuranyl THP tetrahydropyranyl Tos tosylTr trityl

[0326] The following conventional three-letter amino acid abbreviationsare used herein; the conventional one-letter amino acid abbreviationsare NOT used herein: Ala = alanine Arg = arginine Asn = asparagine Asp =aspartic acid Cys = cysteine Gln = glutamine Glu = glutamic acid Gly =glycine His = histidine Ile = isoleucine Leu = leucine Lys = lysine Met= methionine Nle = norleucine Orn = ornithine Phe = phenylalanine Phg =phenylglycine Pro = proline Sar = sarcosine Ser = serine Thr = threonineTrp = tryptophan Tyr = tyrosine Val = valine

[0327] As used herein, the term “bubbles”, as used herein, refers tovesicles which are generally characterized by the presence of one ormore membranes or walls surrounding an internal void that is filled witha gas or precursor thereto. Exemplary bubbles include, for example,liposomes, micelles and the like.

[0328] As used herein, the term “lipid” refers to a synthetic ornaturally-occurring amphipathic compound which comprises a hydrophiliccomponent and a hydrophobic component. Lipids include, for example,fatty acids, neutral fats, phosphatides, glycolipids, aliphatic alcholsand waxes, terpenes and steroids.

[0329] As used herein, the term “lipid composition” refers to acomposition which comprises a lipid compound. Exemplary lipidcompositions include suspensions, emulsions and vesicular compositions.

[0330] As used herein, the term “lipid formulation” refers to acomposition which comprises a lipid compound and a bioactive agent.

[0331] As used herein, the term “vesicle” refers to a spherical entitywhich is characterized by the presence of an internal void. Preferredvesicles are formulated from lipids, including the various lipidsdescribed herein. In any given vesicle, the lipids may be in the form ofa monolayer or bilayer, and the mono- or bilayer lipids may be used toform one of more mono- or bilayers. In the case of more than one mono-or bilayer, the mono- or bilayers are generally concentric. The lipidvesicles described herein include such entities commonly referred to asliposomes, micelles, bubbles, microbubbles, microspheres and the like.Thus, the lipids may be used to form a unilamellar vesicle (comprised ofone monolayer or bilayer), an oligolamellar vesicle (comprised of abouttwo or about three monolayers or bilayers) or a multilamellar vesicle(comprised of more than about three monolayers or bilayers). Theinternal void of the vesicles may be filled with a liquid, including,for example, an aqueous liquid, a gas, a gaseous precursor, and/or asolid or solute material, including, for example, a bioactive agent, asdesired.

[0332] As used herein, the term “vesicular composition” refers to acomposition which is formulate from lipids and which comprises vesicles.

[0333] As used herein, the term “vesicle formulation” refers to acomposition which comprises vesicles and a bioactive agent.

[0334] As used herein, the term “lipsomes” refers to a generallyspherical cluster or aggregate of amphipathic compounds, including lipidcompounds, typically in the form of one or more concentric layers, forexample, bilayers. They may also be referred to herein as lipidvesicles.

[0335] Angiogenesis is the process of formation of new capillary bloodvessels from existing vasculature. It is an important component of avariety of physiological processes including ovulation, embryonicdevelopment, wound repair, and collateral vascular generation in themyocardium. It is also central to a number of pathological conditionssuch as tumor growth and metastasis, diabetic retinopathy, and maculardegeneration. The process begins with the activation of existingvascular endothelial cells in response to a variety of cytokines andgrowth factors. The activated endothelial cells secrete enzymes thatdegrade the basement membrane of the vessels. The endothelial cells thenproliferate and migrate into the extracellular matrix first formingtubules and subsequently new blood vessels.

[0336] Under normal conditions, endothelial cell proliferation is a veryslow process, but it increases for a short period of time duringembryogenesis, ovulation and wound healing. This temporary increase incell turnover is governed by a combination of a number of growthstimulatory factors and growth suppressing factors. In pathologicalangiogenesis, this normal balance is disrupted resulting in continuedincreased endothelial cell proliferation. Some of the pro-angiogenicfactors that have been identified include basic fibroblast growth factor(bFGF), angiogenin, TGF-alpha, TGF-beta, and vascular endothelium growthfactor (VEGF), while interferon-alpha, interferon-beta andthrombospondin are examples of angiogenesis suppressors.

[0337] Angiogenic factors interact with endothelial cell surfacereceptors such as the receptor tyrosine kinases EGFR, FGFR, PDGFR,Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, and Axl.The receptors Flk-1/KDR, neuropilin-1, and Flt-1 recognize VEGF andthese interactions play key roles in VEGF-induced angiogenesis. The Tiesubfamily of receptor tyrosine kinases are also expressed prominentlyduring blood vessel formation.

[0338] The proliferation and migration of endothelial cells in theextracellular matrix is mediated by interaction with a variety of celladhesion molecules. Integrins are a diverse family of heterodimeric cellsurface receptors by which endothelial cells attach to the extracellularmatrix, each other and other cells. Angiogenesis induced by bFGF orTNF-alpha depend on the agency of the integrin avb3, while angiogenesisinduced by VEGF depends on the integrin avb5 (Cheresh et. al., Science,1995, 270, 1500-2). Induction of expression of the integrins a1b1 anda2b1 on the endothelial cell surface is another important mechanism bywhich VEGF promotes angiogenesis (Senger, et. al., Proc. Natl. Acad, SciUSA, 1997, 94, 13612-7).

[0339] The pharmaceuticals of the present invention are comprised of anon-peptide targeting moiety for the vitronectin receptor that isexpressed or upregulated in angiogenic tumor vasculature.

[0340] The ultrasound contrast agents of the present invention comprisea plurality of vitronectin receptor targeting moieties attached to orincorporated into a microbubble of a biocompatible gas, a liquidcarrier, and a surfactant microsphere, further comprising an optionallinking moiety, L_(n), between the targeting moieties and themicrobubble. In this context, the term liquid carrier means aqueoussolution and the term surfactant means any amphiphilic material whichproduces a reduction in interfacial tension in a solution. A list ofsuitable surfactants for forming surfactant microspheres is disclosed inEP0727225A2, herein incorporated by reference. The term surfactantmicrosphere includes nanospheres, liposomes, vesicles and the like. Thebiocompatible gas can be air, or a fluorocarbon, such as a C₃-C₅perfluoroalkane, which provides the difference in echogenicity and thusthe contrast in ultrasound imaging. The gas is encapsulated or containedin the microsphere to which is attached the biodirecting group,optionally via a linking group. The attachment can be covalent, ionic orby van der Waals forces. Specific examples of such contrast agentsinclude lipid encapsulated perfluorocarbons with a plurality of tumorneovasculature receptor binding peptides, polypeptides orpeptidomimetics.

[0341] X-ray contrast agents of the present invention are comprised ofone or more vitronectin receptor targeting moieties attached to one ormore X-ray absorbing or “heavy” atoms of atomic number 20 or greater,further comprising an optional linking moiety, L_(n), between thetargeting moieties and the X-ray absorbing atoms. The frequently usedheavy atom in X-ray contrast agents is iodine. Recently, X-ray contrastagents comprised of metal chelates (Wallace, R., U.S. Pat. No.5,417,959) and polychelates comprised of a plurality of metal ions(Love, D., U.S. Pat. No. 5,679,810) have been disclosed. More recently,multinuclear cluster complexes have been disclosed as X-ray contrastagents (U.S. Pat. No. 5,804,161, PCT WO91/14460, and PCT WO 92/17215).

[0342] MRI contrast agents of the present invention are comprised of oneor more vitronectin receptor targeting moieties attached to one or moreparamagnetic metal ions, further comprising an optional linking moiety,L_(n), between the targeting moieties and the paramagnetic metal ions.The paramagnetic metal ions are present in the form of metal complexesor metal oxide particles. U.S. Pat. Nos. 5,412,148, and 5,760,191,describe examples of chelators for paramagnetic metal ions for use inMRI contrast agents. U.S. Pat. No. 5,801,228, U.S. Pat. No. 5,567,411,and U.S. Pat. No. 5,281,704, describe examples of polychelants usefulfor complexing more than one paramagnetic metal ion for use in MRIcontrast agents. U.S. Pat. No. 5,520,904, describes particulatecompositions comprised of paramagnetic metal ions for use as MRIcontrast agents.

[0343] The pharmaceuticals of the present invention have the formulae,(Q)_(d)-L_(n)-(C_(h)—X), (Q)_(d)-L_(n)-(C_(h)—X¹)_(d′),(Q)_(d)-L_(n)-(X²)_(d″), and (Q)_(d)-L_(n)-(X³), wherein Q represents anon-peptide that binds to a receptor expressed in angiogenic tumorvasculature, d is 1-10, L_(n) represents an optional linking group,C_(h) represents a metal chelator or bonding moiety, X represents aradioisotope, X¹ represents paramagnetic metal ion, X² represents aparamagnetic metal ion or heavy atom containing insoluble solidparticle, d″ is 1-100, and X³ represents a surfactant microsphere of anechogenic gas. The interaction of the non-peptide recognition sequencesof the vitronectin receptor binding portion of the pharmaceuticals withthe α_(v)β₃ receptor results in localization of the pharmaceuticals inangiogenic tumor vasculature, which express the α_(v)β₃ receptor.

[0344] The pharmaceuticals of the present invention can be synthesizedby several approaches. One approach involves the synthesis of thetargeting non-peptide moiety, Q, and direct attachment of one or moremoieties, Q, to one or more metal chelators or bonding moieties, C_(h),or to a paramagnetic metal ion or heavy atom containing solid particle,or to an echogenic gas microbubble. Another approach involves theattachment of one or more moieties, Q, to the linking group, L_(n),which is then attached to one or more metal chelators or bondingmoieties, C_(h), or to a paramagnetic metal ion or heavy atom containingsolid particle, or to an echogenic gas microbubble. Another approachinvolves the synthesis of a non-peptide, Q, bearing a fragment of thelinking group, L_(n), one or more of which are then attached to theremainder of the linking group and then to one or more metal chelatorsor bonding moieties, C_(h), or to a paramagnetic metal ion or heavy atomcontaining solid particle, or to an echogenic gas microbubble.

[0345] The non-peptide vitronectin binding moieties, Q, optionallybearing a linking group, L_(n), or a fragment of the linking group, canbe synthesized using standard synthetic methods known to those skilledin the art. Preferred methods include but are not limited to thosemethods described below.

[0346] The attachment of linking groups, L_(n), to the non-peptides, Q;chelators or bonding units, C_(h), to the non-peptides, Q, or to thelinking groups, L_(n); and non-peptides, bearing a fragment of thelinking group to the remainder of the linking group, in combinationforming the moiety, (Q)_(d)-L_(n),and then to the moiety C_(h); can allbe performed by standard techniques. These include, but are not limitedto, amidation, esterification, alkylation, and the formation of ureas orthioureas. Procedures for performing these attachments can be found inBrinkley, M., Bioconjugate Chemistry 1992, 3(1), which is incorporatedherein by reference.

[0347] A number of methods can be used to attach the non-peptides, Q, toparamagnetic metal ion or heavy atom containing solid particles, X², byone of skill in the art of the surface modification of solid particles.In general, the targeting moiety Q or the combination (Q)_(d)L_(n) isattached to a coupling group that react with a constituent of thesurface of the solid particle. The coupling groups can be any of anumber of silanes which react with surface hydroxyl groups on the solidparticle surface, as described in co-pending U.S. patent applicationSer. No. 09/356,178, and can also include polyphosphonates,polycarboxylates, polyphosphates or mixtures thereof which couple withthe surface of the solid particles, as described in U.S. Pat. No.5,520,904.

[0348] A number of reaction schemes can be used to attach thenon-peptides, Q, to the surfactant microsphere, X³. These areillustrated in following reaction schemes where S_(f) represents asurfactant moiety that forms the surfactant microsphere.

[0349] Acylation Reaction:

[0350] In these reaction schemes, the substituents S_(f) and Q can bereversed as well.

[0351] The linking group L_(n) can serve several roles. First itprovides a spacing group between the metal chelator or bonding moiety,C_(h), the paramagnetic metal ion or heavy atom containing solidparticle, X², and the surfactant microsphere, X³, and the one or more ofthe non-peptides, Q, so as to minimize the possibility that the moietiesC_(h)—X, C_(h)—X¹, X², and X³, will interfere with the interaction ofthe recognition sequences of Q with angiogenic tumor vasculaturereceptors. The necessity of incorporating a linking group in a reagentis dependent on the identity of Q, C_(h)—X, C_(h)—X¹, X², and X³. IfC_(h)—X, C_(h)—X¹, X², and X³, cannot be attached to Q withoutsubstantially diminishing its affinity for the receptors, then a linkinggroup is used. A linking group also provides a means of independentlyattaching multiple non-pepetides, Q, to one group that is attached toC_(h)—X, C_(h)—X¹, X², or X³.

[0352] The linking group also provides a means of incorporating apharmacokinetic modifier into the pharmaceuticals of the presentinvention. The pharmacokinetic modifier serves to direct thebiodistibution of the injected pharmaceutical other than by theinteraction of the targeting moieties, Q, with the vitronectin receptorsexpressed in the tumor neovasculature. A wide variety of functionalgroups can serve as pharmacokinetic modifiers, including, but notlimited to, carbohydrates, polyalkylene glycols, peptides or otherpolyamino acids, and cyclodextrins. The modifiers can be used to enhanceor decrease hydrophilicity and to enhance or decrease the rate of bloodclearance. The modifiers can also be used to direct the route ofelimination of the pharmaceuticals. Preferred pharmacokinetic modifiersare those that result in moderate to fast blood clearance and enhancedrenal excretion.

[0353] The metal chelator or bonding moiety, C_(h), is selected to formstable complexes with the metal ion chosen for the particularapplication. Chelators or bonding moieties for diagnosticradiopharmaceuticals are selected to form stable complexes with theradioisotopes that have imageable gamma ray or positron emissions, suchas ^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁰Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y.

[0354] Chelators for technetium, copper and gallium isotopes areselected from diaminedithiols, monoamine-monoamidedithiols,triamide-monothiols, monoamine-diamide-monothiols, diaminedioximes, andhydrazines. The chelators are generally tetradentate with donor atomsselected from nitrogen, oxygen and sulfur. Preferred reagents arecomprised of chelators having amine nitrogen and thiol sulfur donoratoms and hydrazine bonding units. The thiol sulfur atoms and thehydrazines may bear a protecting group which can be displaced eitherprior to using the reagent to synthesize a radiopharmaceutical orpreferably in situ during the synthesis of the radiopharmaceutical.

[0355] Exemplary thiol protecting groups include those listed in Greeneand Wuts, “Protective Groups in Organic Synthesis” John Wiley & Sons,New York (1991), the disclosure of which is hereby incorporated byreference. Any thiol protecting group known in the art can be used.Examples of thiol protecting groups include, but are not limited to, thefollowing: acetamidomethyl, benzamidomethyl, 1-ethoxyethyl, benzoyl, andtriphenylmethyl.

[0356] Exemplary protecting groups for hydrazine bonding units arehydrazones which can be aldehyde or ketone hydrazones havingsubstituents selected from hydrogen, alkyl, aryl and heterocycle.Particularly preferred hydrazones are described in co-pending U.S. Ser.No. 08/476,296 the disclosure of which is herein incorporated byreference in its entirety.

[0357] The hydrazine bonding unit when bound to a metal radionuclide istermed a hydrazido, or diazenido group and serves as the point ofattachment of the radionuclide to the remainder of theradiopharmaceutical. A diazenido group can be either terminal (only oneatom of the group is bound to the radionuclide) or chelating. In orderto have a chelating diazenido group at least one other atom of the groupmust also be bound to the radionuclide. The atoms bound to the metal aretermed donor atoms.

[0358] Chelators for ¹¹¹In and ⁸⁶Y are selected from cyclic and acyclicpolyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazazcyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.Procedures for synthesizing these chelators that are not commerciallyavailable can be found in Brechbiel, M. and Gansow, O., J. Chem. Soc.Perkin Trans. 1992, 1, 1175; Brechbiel, M. and Gansow, O., BioconjugateChem. 1991, 2, 187; Deshpande, S., et. al., J. Nucl. Med. 1990, 31, 473;Kruper, J., U.S. Pat. No. 5,064,956, and Toner, J., U.S. Pat. No.4,859,777, the disclosures of which are hereby incorporated by referencein their entirety.

[0359] The coordination sphere of metal ion includes all the ligands orgroups bound to the metal. For a transition metal radionuclide to bestable it typically has a coordination number (number of donor atoms)comprised of an integer greater than or equal to 4 and less than orequal to 8; that is there are 4 to 8 atoms bound to the metal and it issaid to have a complete coordination sphere. The requisite coordinationnumber for a stable radionuclide complex is determined by the identityof the radionuclide, its oxidation state, and the type of donor atoms.If the chelator or bonding unit does not provide all of the atomsnecessary to stabilize the metal radionuclide by completing itscoordination sphere, the coordination sphere is completed by donor atomsfrom other ligands, termed ancillary or co-ligands, which can also beeither terminal or chelating.

[0360] A large number of ligands can serve as ancillary or co-ligands,the choice of which is determined by a variety of considerations such asthe ease of synthesis of the radiopharmaceutical, the chemical andphysical properties of the ancillary ligand, the rate of formation, theyield, and the number of isomeric forms of the resultingradiopharmaceuticals, the ability to administer said ancillary orco-ligand to a patient without adverse physiological consequences tosaid patient, and the compatibility of the ligand in a lyophilized kitformulation. The charge and lipophilicity of the ancillary ligand willeffect the charge and lipophilicity of the radiopharmaceuticals. Forexample, the use of 4,5-dihydroxy-1,3-benzene disulfonate results inradiopharmaceuticals with an additional two anionic groups because thesulfonate groups will be anionic under physiological conditions. The useof N-alkyl substituted 3,4-hydroxypyridinones results inradiopharmaceuticals with varying degrees of lipophilicity depending onthe size of the alkyl substituents.

[0361] Preferred technetium radiopharmaceuticals of the presentinvention are comprised of a hydrazido or diazenido bonding unit and anancillary ligand, A_(L1), or a bonding unit and two types of ancillaryA_(L1) and A_(L2), or a tetradentate chelator comprised of two nitrogenand two sulfur atoms. Ancillary ligands A_(L1) are comprised of two ormore hard donor atoms such as oxygen and amine nitrogen (sp³hybridized). The donor atoms occupy at least two of the sites in thecoordination sphere of the radionuclide metal; the ancillary ligandA_(L1) serves as one of the three ligands in the ternary ligand system.Examples of ancillary ligands A_(L1) include but are not limited todioxygen ligands and functionalized aminocarboxylates. A large number ofsuch ligands are available from commercial sources.

[0362] Ancillary dioxygen ligands include ligands that coordinate to themetal ion through at least two oxygen donor atoms. Examples include butare not limited to: glucoheptonate, gluconate, 2-hydroxyisobutyrate,lactate, tartrate, mannitol, glucarate, maltol, Kojic acid,2,2-bis(hydroxymethyl)propionic acid, 4,5-dihydroxy-1,3-benzenedisulfonate, or substituted or unsubstituted 1,2 or 3,4hydroxypyridinones. (The names for the ligands in these examples referto either the protonated or non-protonated forms of the ligands.)

[0363] Functionalized aminocarboxylates include ligands that have acombination of amine nitrogen and oxygen donor atoms. Examples includebut are not limited to: iminodiacetic acid, 2,3-diaminopropionic acid,nitrilotriacetic acid, N,N′-ethylenediamine diacetic acid,N,N,N′-ethylenediamine triacetic acid, hydroxyethylethylenediaminetriacetic acid, and N,N′-ethylenediamine bis-hydroxyphenylglycine. (Thenames for the ligands in these examples refer to either the protonatedor non-protonated forms of the ligands.)

[0364] A series of functionalized aminocarboxylates are disclosed byBridger et. al. in U.S. Pat. No. 5,350,837, herein incorporated byreference, that result in improved rates of formation of technetiumlabeled hydrazino modified proteins. We have determined that certain ofthese aminocarboxylates result in improved yields of theradiopharmaceuticals of the present invention. The preferred ancillaryligands A_(L1) functionalized aminocarboxylates that are derivatives ofglycine; the most preferred is tricine(tris(hydroxymethyl)methylglycine).

[0365] The most preferred technetium radiopharmaceuticals of the presentinvention are comprised of a hydrazido or diazenido bonding unit and twotypes of ancillary designated A_(L1) and A_(L2), or a diaminedithiolchelator. The second type of ancillary ligands A_(L2) are comprised ofone or more soft donor atoms selected from the group: phosphinephosphorus, arsine arsenic, imine nitrogen (sp² hybridized), sulfur (sp²hybridized) and carbon (sp hybridized); atoms which have p-acidcharacter. Ligands A_(L2) can be monodentate, bidentate or tridentate,the denticity is defined by the number of donor atoms in the ligand. Oneof the two donor atoms in a bidentate ligand and one of the three donoratoms in a tridentate ligand must be a soft donor atom. We havedisclosed in co-pending U.S. Ser. No. 08/415,908, and U.S. Ser. Nos.60/013360 and 08/646,886, the disclosures of which are hereinincorporated by reference in their entirety, that radiopharmaceuticalscomprised of one or more ancillary or co-ligands A_(L2) are more stablecompared to radiopharmaceuticals that are not comprised of one or moreancillary ligands, A_(L2); that is, they have a minimal number ofisomeric forms, the relative ratios of which do not change significantlywith time, and that remain substantially intact upon dilution.

[0366] The ligands A_(L2) that are comprised of phosphine or arsinedonor atoms are trisubstituted phosphines, trisubstituted arsines,tetrasubstituted diphosphines and tetrasubstituted diarsines. Theligands A_(L2) that are comprised of imine nitrogen are unsaturated oraromatic nitrogen-containing, 5 or 6-membered heterocycles. The ligandsthat are comprised of sulfur (sp² hybridized) donor atoms arethiocarbonyls, comprised of the moiety C═S. The ligands comprised ofcarbon (sp hybridized) donor atoms are isonitriles, comprised of themoiety CNR, where R is an organic radical. A large number of suchligands are available from commercial sources. Isonitriles can besynthesized as described in European Patent 0107734 and in U.S. Pat. No.4,988,827, herein incorporated by reference.

[0367] Preferred ancillary ligands A_(L2) are trisubstituted phosphinesand unsaturated or aromatic 5 or 6 membered heterocycles. The mostpreferred ancillary ligands A_(L2) are trisubstituted phosphines andunsaturated 5 membered heterocycles.

[0368] The ancillary ligands A_(L2) may be substituted with alkyl, aryl,alkoxy, heterocycle, aralkyl, alkaryl and arylalkaryl groups and may ormay not bear functional groups comprised of heteroatoms such as oxygen,nitrogen, phosphorus or sulfur. Examples of such functional groupsinclude but are not limited to: hydroxyl, carboxyl, carboxamide, nitro,ether, ketone, amino, ammonium, sulfonate, sulfonamide, phosphonate, andphosphonamide. The functional groups may be chosen to alter thelipophilicity and water solubility of the ligands which may affect thebiological properties of the radiopharmaceuticals, such as altering thedistribution into non-target tissues, cells or fluids, and the mechanismand rate of elimination from the body.

[0369] Chelators or bonding moieties for therapeuticradiopharmaceuticals are selected to form stable complexes with theradioisotopes that have alpha particle, beta particle, Auger orCoster-Kronig electron emissions, such as ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho,¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au,¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir. Chelators forrhenium, copper, palladium, platinum, iridium, rhodium, silver and goldisotopes are selected from diaminedithiols, monoamine-monoamidedithiols,triamide-monothiols, monoamine-diamide-monothiols, diaminedioximes, andhydrazines. Chelators for yttrium, bismuth, and the lanthanide isotopesare selected from cyclic and acyclic polyaminocarboxylates such as DTPA,DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

[0370] Chelators for magnetic resonance imaging contrast agents areselected to form stable complexes with paramagnetic metal ions, such asGd(III), Dy(III), Fe(III), and Mn(II), are selected from cyclic andacyclic polyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

[0371] The technetium and rhenium radiopharmaceuticals of the presentinvention comprised of a hydrazido or diazenido bonding unit can beeasily prepared by admixing a salt of a radionuclide, a reagent of thepresent invention, an ancillary ligand A_(L1), an ancillary ligandA_(L2), and a reducing agent, in an aqueous solution at temperaturesfrom 0 to 100° C. The technetium and rhenium radiopharmaceuticals of thepresent invention comprised of a tetradentate chelator having twonitrogen and two sulfur atoms can be easily prepared by admixing a saltof a radionuclide, a reagent of the present invention, and a reducingagent, in an aqueous solution at temperatures from 0 to 100° C.

[0372] When the bonding unit in the reagent of the present invention ispresent as a hydrazone group, then it must first be converted to ahydrazine, which may or may not be protonated, prior to complexationwith the metal radionuclide. The conversion of the hydrazone group tothe hydrazine can occur either prior to reaction with the radionuclide,in which case the radionuclide and the ancillary or co-ligand or ligandsare combined not with the reagent but with a hydrolyzed form of thereagent bearing the chelator or bonding unit, or in the presence of theradionuclide in which case the reagent itself is combined with theradionuclide and the ancillary or co-ligand or ligands. In the lattercase, the pH of the reaction mixture must be neutral or acidic.

[0373] Alternatively, the radiopharmaceuticals of the present inventioncomprised of a hydrazido or diazenido bonding unit can be prepared byfirst admixing a salt of a radionuclide, an ancillary ligand A_(L1), anda reducing agent in an aqueous solution at temperatures from 0 to 100°C. to form an intermediate radionuclide complex with the ancillaryligand A_(L1) then adding a reagent of the present invention and anancillary ligand A_(L2) and reacting further at temperatures from 0 to100° C.

[0374] Alternatively, the radiopharmaceuticals of the present inventioncomprised of a hydrazido or diazenido bonding unit can be prepared byfirst admixing a salt of a radionuclide, an ancillary ligand A_(L1), areagent of the present invention, and a reducing agent in an aqueoussolution at temperatures from 0 to 100° C. to form an intermediateradionuclide complex, and then adding an ancillary ligand A_(L2) andreacting further at temperatures from 0 to 100° C.

[0375] The technetium and rhenium radionuclides are preferably in thechemical form of pertechnetate or perrhenate and a pharmaceuticallyacceptable cation. The pertechnetate salt form is preferably sodiumpertechnetate such as obtained from commercial Tc-99m generators. Theamount of pertechnetate used to prepare the radiopharmaceuticals of thepresent invention can range from 0.1 mCi to 1 Ci, or more preferablyfrom 1 to 200 mCi.

[0376] The amount of the reagent of the present invention used toprepare the technetium and rhenium radiopharmaceuticals of the presentinvention can range from 0.01 μg to 10 mg, or more preferably from 0.5μg to 200 μg. The amount used will be dictated by the amounts of theother reactants and the identity of the radiopharmaceuticals of thepresent invention to be prepared.

[0377] The amounts of the ancillary ligands A_(L1) used can range from0.1 mg to 1 g, or more preferably from 1 mg to 100 mg. The exact amountfor a particular radiopharmaceutical is a function of identity of theradiopharmaceuticals of the present invention to be prepared, theprocedure used and the amounts and identities of the other reactants.Too large an amount of A_(L1) will result in the formation ofby-products comprised of technetium labeled A_(L1) without abiologically active molecule or by-products comprised of technetiumlabeled biologically active molecules with the ancillary ligand A_(L1)but without the ancillary ligand A_(L2). Too small an amount of A_(L1)will result in other by-products such as technetium labeled biologicallyactive molecules with the ancillary ligand A_(L2) but without theancillary ligand A_(L1), or reduced hydrolyzed technetium, or technetiumcolloid.

[0378] The amounts of the ancillary, ligands A_(L2) used can range from0.001 mg to 1 g, or more preferably from 0.01 mg to 10 mg. The exactamount for a particular radiopharmaceutical is a function of theidentity of the radiopharmaceuticals of the present invention to beprepared, the procedure used and the amounts and identities of the otherreactants. Too large an amount of A_(L2) will result in the formation ofby-products comprised of technetium labeled A_(L2) without abiologically active molecule or by-products comprised of technetiumlabeled biologically active molecules with the ancillary ligand A_(L2)but without the ancillary ligand A_(L1). If the reagent bears one ormore substituents that are comprised of a soft donor atom, as definedabove, at least a ten-fold molar excess of the ancillary ligand A_(L2)to the reagent of formula 2 is required to prevent the substituent frominterfering with the coordination of the ancillary ligand A_(L2) to themetal radionuclide.

[0379] Suitable reducing agents for the synthesis of theradiopharmaceuticals of the present invention include stannous salts,dithionite or bisulfite salts, borohydride salts, andformamidinesulfinic acid, wherein the salts are of any pharmaceuticallyacceptable form. The preferred reducing agent is a stannous salt. Theamount of a reducing agent used can range from 0.001 mg to 10 mg, ormore preferably from 0.005 mg to 1 mg.

[0380] The specific structure of a radiopharmaceutical of the presentinvention comprised of a hydrazido or diazenido bonding unit will dependon the identity of the reagent of the present invention used, theidentity of any ancillary ligand A_(L1), the identity of any ancillaryligand A_(L2), and the identity of the radionuclide.Radiopharmaceuticals comprised of a hydrazido or diazenido bonding unitsynthesized using concentrations of reagents of<100 μg/mL, will becomprised of one hydrazido or diazenido group. Those synthesized using>1mg/mL concentrations will be comprised of two hydrazido or diazenidogroups from two reagent molecules. For most applications, only a limitedamount of the biologically active molecule can be injected and notresult in undesired side-effects, such as chemical toxicity,interference with a biological process or an altered biodistribution ofthe radiopharmaceutical. Therefore, the radiopharmaceuticals whichrequire higher concentrations of the reagents comprised in part of thebiologically active molecule, will have to be diluted or purified aftersynthesis to avoid such side-effects.

[0381] The identities and amounts used of the ancillary ligands A_(L1)and A_(L2) will determine the values of the variables y and z. Thevalues of y and z can independently be an integer from 1 to 2. Incombination, the values of y and z will result in a technetiumcoordination sphere that is made up of at least five and no more thanseven donor atoms. For monodentate ancillary ligands A_(L2), z can be aninteger from 1 to 2; for bidentate or tridentate ancillary ligandsA_(L2), z is 1. The preferred combination for monodentate ligands is yequal to 1 or 2 and z equal to 1. The preferred combination forbidentate or tridentate ligands is y equal to 1 and z equal to 1.

[0382] The indium, copper, gallium, silver, palladium, rhodium, gold,platinum, bismuth, yttrium and lanthanide radiopharmaceuticals of thepresent invention can be easily prepared by admixing a salt of aradionuclide and a reagent of the present invention, in an aqueoussolution at temperatures from 0 to 100° C. These radionuclides aretypically obtained as a dilute aqueous solution in a mineral acid, suchas hydrochloric, nitric or sulfuric acid. The radionuclides are combinedwith from one to about one thousand equivalents of the reagents of thepresent invention dissolved in aqueous solution. A buffer is typicallyused to maintain the pH of the reaction mixture between 3 and 10.

[0383] The gadolinium, dysprosium, iron and manganesemetallopharmaceuticals of the present invention can be easily preparedby admixing a salt of the paramagnetic metal ion and a reagent of thepresent invention, in an aqueous solution at temperatures from 0 to 100°C. These paramagnetic metal ions are typically obtained as a diluteaqueous solution in a mineral acid, such as hydrochloric, nitric orsulfuric acid. The paramagnetic metal ions are combined with from one toabout one thousand equivalents of the reagents of the present inventiondissolved in aqueous solution. A buffer is typically used to maintainthe pH of the reaction mixture between 3 and 10.

[0384] The total time of preparation will vary depending on the identityof the metal ion, the identities and amounts of the reactants and theprocedure used for the preparation. The preparations may be complete,resulting in>80% yield of the radiopharmaceutical, in 1 minute or mayrequire more time. If higher purity metallopharmaceuticals are needed ordesired, the products can be purified by any of a number of techniqueswell known to those skilled in the art such as liquid chromatography,solid phase extraction, solvent extraction, dialysis or ultrafiltration.

[0385] Buffers useful in the preparation of metallopharmaceuticals andin diagnostic kits useful for the preparation of saidradiopharmaceuticals include but are not limited to phosphate, citrate,sulfosalicylate, and acetate. A more complete list can be found in theUnited States Pharmacopeia.

[0386] Lyophilization aids useful in the preparation of diagnostic kitsuseful for the preparation of radiopharmaceuticals include but are notlimited to mannitol, lactose, sorbitol, dextran, Ficoll, andpolyvinylpyrrolidine(PVP).

[0387] Stabilization aids useful in the preparation ofmetallopharmaceuticals and in diagnostic kits useful for the preparationof radiopharmaceuticals include but are not limited to ascorbic acid,cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite,gentisic acid, and inositol.

[0388] Solubilization aids useful in the preparation ofmetallopharmaceuticals and in diagnostic kits useful for the preparationof radiopharmaceuticals include but are not limited to ethanol,glycerin, polyethylene glycol, propylene glycol, polyoxyethylenesorbitan monooleate, sorbitan monoloeate, polysorbates,poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers(Pluronics) and lecithin. Preferred solubilizing aids are polyethyleneglycol, and Pluronics.

[0389] Bacteriostats useful in the preparation of metallopharmaceuticalsand in diagnostic kits useful for the preparation ofradiopharmaceuticals include but are not limited to benzyl alcohol,benzalkonium chloride, chlorbutanol, and methyl, propyl or butylparaben.

[0390] A component in a diagnostic kit can also serve more than onefunction. A reducing agent can also serve as a stabilization aid, abuffer can also serve as a transfer ligand, a lyophilization aid canalso serve as a transfer, ancillary or co-ligand and so forth.

[0391] The diagnostic radiopharmaceuticals are administered byintravenous injection, usually in saline solution, at a dose of 1 to 100mCi per 70 kg body weight, or preferably at a dose of 5 to 50 mCi.Imaging is performed using known procedures.

[0392] The therapeutic radiopharmaceuticals are administered byintravenous injection, usually in saline solution, at a dose of 0.1 to100 mCi per 70 kg body weight, or preferably at a dose of 0.5 to 5 mCiper 70 kg body weight.

[0393] The magnetic resonance imaging contrast agents of the presentinvention may be used in a similar manner as other MRI agents asdescribed in U.S. Pat. No. 5,155,215; U.S. Pat. No. 5,087,440;Margerstadt et al., Magn. Reson. Med., 1986, 3, 808; Runge et al.,Radiology, 1988, 166, 835; and Bousquet et al., Radiology, 1988, 166,693. Generally, sterile aqueous solutions of the contrast agents areadministered to a patient intravenously in dosages ranging from 0.01 to1.0 mmoles per kg body weight.

[0394] For use as X-ray contrast agents, the compositions of the presentinvention should generally have a heavy atom concentration of 1 mM to 5M, preferably 0.1 M to 2 M. Dosages, administered by intravenousinjection, will typically range from 0.5 mmol/kg to 1.5 mmol/kg,preferably 0.8 mmol/kg to 1.2 mmol/kg. Imaging is performed using knowntechniques, preferably X-ray computed tomography.

[0395] The ultrasound contrast agents of the present invention areadministered by intravenous injection in an amount of 10 to 30 μL of theechogenic gas per kg body weight or by infusion at a rate ofapproximately 3 μL/kg/min. Imaging is performed using known techniquesof sonography.

[0396] Other features of the invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention and are not intended to belimiting thereof.

EXAMPLES

[0397] Representative materials and methods that may be used inpreparing the compounds of the invention are described further below.

[0398] Manual solid phase peptide synthesis was performed in 25 mLpolypropylene filtration tubes purchased from BioRad Inc., or in 60 mLhour-glass reaction vessels purchased from Peptides International. Oximeresin (substitution level=0.96 mmol/g) was prepared according topublished procedure (DeGrado and Kaiser, J. Org. Chem. 1980, 45, 1295),or was purchased from Novabiochem (substitution level=0.62 mmol/g). Allchemicals and solvents (reagent grade) were used as supplied from thevendors cited without further purification. t-Butyloxycarbonyl (Boc)amino acids and other starting amino acids may be obtained commerciallyfrom Bachem Inc., Bachem Biosciences Inc. (Philadelphia, Pa.), AdvancedChemTech (Louisville, Ky.), Peninsula Laboratories (Belmont, Calif.), orSigma (St. Louis, Mo.).2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) and TBTU were purchased from Advanced ChemTech.N-methylmorpholine (NMM), m-cresol, D-2-aminobutyric acid (Abu),trimethylacetylchloride, diisopropylethylamine (DIEA), 1,2,4-triazole,stannous chloride dihydrate, and tris(3-sulfonatophenyl)phosphinetrisodium salt (TPPTS) were purchased from Aldrich Chemical Company.Bis(3-sulfonatophenyl)phenylphosphine disodium salt (TPPDS) was preparedby the published procedure (Kuntz, E., U.S. Pat. No. 4,248,802).(3-Sulfonatophenyl)diphenylphosphine monosodium salt (TPPMS)waspurchased from TCI America, Inc. Tricine was obtained from ResearchOrganics, Inc. Technetium-99m-pertechnetate (^(99m)TcO₄ ⁻) was obtainedfrom a DuPont Pharma ⁹⁹Mo/^(99m)Tc Technelite® generator.In-111-chloride (Indichlor®) was obtained from Amersham Medi-Physics,Inc. Sm-153-chloride and Lutetium-177-chloride were obtained from theUniversity of Missouri Research Reactor (MURR). Yttrium-90 chloride wasobtained from the Pacific Northwest Research Laboratories.Dimethylformamide (DMF), ethyl acetate, chloroform (CHCl₃), methanol(MeOH), pyridine and hydrochloric acid (HCl) were obtained from Baker.Acetonitrile, dichloromethane (DCM), acetic acid (HOAc), trifluoroaceticacid (TFA), ethyl ether, triethylamine, acetone, and magnesium sulfatewere commercially obtained. Absolute ethanol was obtained from QuantumChemical Corporation.

[0399] Synthesis of Boc-Glu-(OTFP)-OTFP

[0400] To a solution of Boc-Glu-OH (28.9 g, 117 mmol) in DMF (500 mL) atroom temperature, and under nitrogen, was added a solution of2,3,5,6-tetrafluorophenol (48.2 g, 290 mmol) in DMF (50 mL). Afterstirring for 10 min. EDC (55.6 g, 290 mmol) was added and the reactionmixture was stirred for about 96 h. The volatiles were removed in vacuoand the residue was triturated in 0.1 N HCl (750 mL). To this mixturewas added ethyl acetate (600 mL), the layers separated. The aqueouslayer was extracted with ethyl acetate (3×˜500 mL), and all the ethylacetate fractions were combined, washed with water (300 mL) and brine(300 mL), dried (MgSO₄), and concentrated to give a tan solid (62 g).The tan solid was washed with acetonitrile to give the title compound(45.5 g, 73%) in purified form.

[0401] ESMS: Calculated for C₂₂H₁₇F₈NO₆, 543.09; found, 566.0 [M+Na]⁺¹.

Example 1 Preparation of(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoyl amino)butanoic Acid

[0402]

[0403] Step 1A. Synthesis of tert-butyl 3-(((3-((tert-butoxy)carbonylamino)propyl)methylamino)methyl)-4-fluorobenzoate

[0404] Crude tert-butyl-4-fluoro-3(alpha-bromomethyl)benzoate (4.6 g.,16 mmol), prepared as described in (WO 95/18619, PCT/US95/00248), wasdissolved in 100 mL THF, along with3-tert-butoxycarbonylamino-1-propylamine hydrochloride (2.9 g., 16.6mmol) and diisopropylethylamine added (4.6 g., 36 mmol). The solutionwas stirred overnight, diluted with 1N NaOH, and extracted with threeportions of ether. The combined organics were washed with water and sat.NaCl, dried over MgSO₄, and concentrated under vacuum to 5.7 g. of ayellow oil. This was purified by flash chromatography (CH₂Cl₂/EtOAc) toafford the product as a clear oil (2.04 g., ˜35%). ¹HNMR (600 MHz,DMSO-d6): 7.99 (dd, J=2, 5.1 Hz, 1H) , 7.78 (ddd, J=2.3, 2.8, 3.0 Hz,1H), 7.22 (dd, J=8.8, 0.7, 1H), 6.73 (b, 1H), 3.68 (s, 2H), 2.94 (m,2H), 2.15 (b, 1H), 1.51 (s, 9H), 1.49 (m, 2H), 1.33 (s, 9H); MS (ES):765.4 [2M+H]⁺, 383.3 [M+H]⁺.

[0405] Step 1B. Synthesis of methyl (S)-3-N-(3-((tert-butoxyl)carbonylamino)propyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate

[0406] The product of Step A (2 g, 5.3 mmol) was dissolved in 20 mL dryDMF, along with N-Cbz-L-aspartic acid β-methyl ester (1.65 g, 5.9 mmol),and 1-hydroxybenzotriazole hydrate (800 mg, 5.9 mmol) under a nitrogenatmosphere. Dicyclohexylcarbodiimide (1M in CH₂Cl₂, 5.9 mL, 5.9 mmol)was added via syringe, and the solution stirred 18 hr.

[0407] Ether (25 mL) was added and the solids were filtered and rinsedwith ether. The filtrate was concentrated, redissolved in ether,filtered, and the filtrate washed with sat. bicarbonate, water, and sat.NaCl. It was dried (Na2SO4), filtered and concentrated to a yellow oilwhich was purified by flash chromatography (4:1 CH2Cl2/EtOAc) to affordthe product (3.0 g, 87%) as a clear oil. ¹HNMR (600 MHz, DMSO-d6):mixture of amide rotamers: 7.82 (m, 2H), 7.71 (m, 1H), 7.3 (m,6H), 6.72(bd, 1H), 5.02 (dd, J=12.5, 25.7 Hz, 1H), 4.44-4.88 (m, 4H), 3.52 (d,2H), 3.27 (d, 3H), 3.10-3.45 (m, 4H)2.45 -2.90 (m, 4H), 1.55 (m, 2H),1.49 (s, 9H), 1.31 (s, 9H); MS-ES: 590.3 [(M-tBu)+H]⁺, 646.4 [M+H]⁺,668.4 [M+Na]⁺.

[0408] Step 1C: Synthesis of methyl(S)-3-amino-3-(N-(3-((tert-butoxy)carbonylamino)propyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)propanoate

[0409] The product of step B (2.8 g, 4.4 mmol) was dissolved in MeOH (50mL) with 10% Pd/C (530 mg) and shaken under a hydrogen atmosphere (50psi) in a Parr shaker for 2 hr. The reaction mixture was filteredthrough Celite® and concentrated to a clear oil (2.14 g, 94%) undervacuum, which was not further purified. MS-ES: 512.4 [M+H]⁺, 1023.5[2M+H]⁺;

[0410] Step 1D: Synthesis of methyl (S)-2-(2,5-diaza-9-((tert-butyl)oxycarbonyl)-5-(3-((tert-butoxy)carbonylamino)propyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0411] The crude oil from C (2.14 g, 4.0 mmol) was dissolved in dryN-methylpyrollidinone (50 mL) along with 2,6-di-tert-butylpyridine (2.1mL, 9.2 mmol) under nitrogen. The solution was heated at 125° C. in anoil bath for 43 hours. The solution was cooled, poured into 100 mLwater, and extracted with ethyl acetate. The organics were concentratedto an oil and purified by flash chromatography (CH₂Cl₂/EtOAc) to afford1.0 g (46%) of the product. MS-ES: 392.3 [(M-tBoc)+H]⁺436.3[(M-tBu)+H]⁺492.4 [M+H]⁺, 983.6 [2M+H]⁺;

[0412] Step 1E: Synthesis of (S)-2,5-diaza-5-(3-((tert-butoxy)carbonylamino)propyl)-3-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-9-carboxylicacid

[0413] The ester from D (880 mg, 1.8 mmol) was dissolved indichloromethane (12 mL) and trifluoroacetic acid (6 mL) added withstirring under nitrogen. The reaction was stirred 2 hours, concentratedunder vacuum, and redissolved in 7 mL dichloromethane. Acetonitrile (7mL) was added, followed by di-tert-butyldicarbonate (590 mg, 2.7 mmol)and diisopropylethylamine (1.4 mL, 7.6 mmol). The reaction was stirredovernight under nitrogen. EtOAc (15 mL) was added and the entiresolution was washed with 5% citric acid and brine, dried (MgSO4), andconcentrated to 1.12 g of oil. This was purified by flash chromatography(CH2Cl2/EtOAc/MeOH) and the residue dissolved in 0.1% TFA/acetonitrile(50 mL) and lyophilized to afford the product (680 mg, 69%) as a whitepowder. ¹HNMR (600 MHz, DMSO-d6): 12.14 (b, 1H), 7.62 (d, J=1.8 Hz, 1H),7.53 (dd, J=1.9 Hz, 8.5 Hz, 1H), 6.66 (bt, J=5.4 Hz, 1H), 6.56 (d, J=8.5Hz, 1H), 6.55 (m, 1H) 5.41 (d, J=16.6 Hz)1H), 5.15 (dd, J=5 Hz, 8.8 Hz,1H), 4.02 (d, 16.7 Hz, 1H), 3.60 (s, 3H), 3.38 (m, 2H), 2.84 (m, 2H),2.82 (dd, J=8.8 Hz, 16.6 Hz, 1H), 2.67 (dd, J=5.3Hz, 16.6 Hz, 1H), 1.50(m, 2H), 1.36 (s, 9H); LRMS(ES): 380.3 [(M-tBu)+H]⁺, 436.3 [M+H]⁺.

[0414] Step 1F. Synthesis of methyl(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(3-((tert-butoxy)carbonylamino)propyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0415] The product of step 1E (476 mg, 1.09 mmol) was dissolved in drydimethylformamide along with 2-(methylaminomethyl)benzimidazoledihydrochloride (290 mg, 1.25 mmol, prepared according to F. Ali et.al., WO 96/00730), hydroxybenzotriazole hydrate (HOBT) (154 mg, 1.14mmol), ethyl dimethylaminopropylcarbodiimide hydrochloride (261 mg, 1.36mmol), and diisopropylethylamine (1.1 mL, 6 mmol). The solution wasstirred for 23 hr under nitrogen and then concentrated. The residue waspartitioned with ethyl acetate/water, and the aqueous layer extractedwith 2 portions of ethyl acetate. The combined organic layers werewashed with water and brine and concentrated. The residue was purifiedby flash chromatography on silica (95:5 ethyl acetate/methanol) and theproduct fractions concentrated to afford the product (435 mg, 69%) as acrunchy foam after drying under vacuum. LRMS(ES): 579.4 [(M+H]⁺. ¹HNMR(600.1300 MHz, DMSO-d6): 12.34 (b, 1H), 7.58 (d, J=1.8 Hz, 1H), 7.48(dd, J=1.9 Hz, 8.5 Hz, 1H), 7.24 (s, 1H), 7.17 (m, 3H), 6.64 (t, 1H),6.56 (d, 1H), 6.55 (m, 1H) 6.21 (s, 1H), 5.41 (d, J=16.6 Hz, 1H), 5.10(dd, J=5 Hz, 8.8 Hz, 1H), 4.76 (q, 2H), 3.89 (d, 16.6 Hz, 1H), 3.60 (s,3H), 3.37 (m, 2H), 3.04 (s, 3H), 2.82 (m, 3H), 2.64 (dd, J=5.3Hz, 16.6Hz, 1H), 1.48 (m, 2H), 1.34 (s, 9H).

[0416] Step 1G: Synthesis of(S,S)-7-((tert-butyl)oxycarbonyl)-2-(2-((tert-butyl)oxycarbonyl)ethyl)-3-oxo-5-((phenylmethoxy)carbonylamino)carbonyl)heptanoic acid

[0417] Gamma-tert-butoxy-Z-glutamic acid succinimide ester (2.0 g, 4.75mmol) was dissolved in dimethylformamide, and gamma-tert-butoxyglutamicacid (0.98 g, 4.8 mmol) added, followed by diisopropylethylamine (1.75mL, 10.1 mmol). The solution was stirred 18 hr, concentrated, and theresidue partitioned into ethyl acetate/10% citric acid. The aqueousfraction was extracted with ethyl acetate and the combined organics werewashed with water, 10% potassium hydrogen sulfate, and brine, and thenconcentrated. The residual oil was purified by flash chromatography onsilica (CH₂Cl₂/EtOAc/EtOH, 1:1:0.5%) and the product fractions combinedand evaporated to yield the product (1.3 g, 53%) as a gummy solid. LRMS(ES): 523.4 [M+H]⁺, 467.4; ¹HNMR (600.1330 MHz, CDCl₃) 7.30 (m, 6H),5.80 (d, 1H), 5.09 (m, 2H), 4.53 (m, 1H), 4.29 (m, 1H), 2.36 (m, 4H),1.88-2.16 (m, 4H), 1.42 (s, 9 H), 1.41 (s, 9H).

[0418] Step 1H: Synthesis of tert-butyl(S,S,S)-4-(N-(3-(3,6-diaza-5-((methoxycarbonyl)methyl)-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxycarbonylamino)butanoylamino)butanoate

[0419] The product of 1F (40 mg, 70 μmol) was dissolved indichloromethane (1 mL) under nitrogen. To this was added triethylsilane(110 uL, 0.7 mmol) and trifluoroacetic acid (1 mL). The reaction wasstirred 60 min, concentrated, and reconcentrated with 5 mL toluene. Theresidue was dissolved in dry dimethylformamide (1 mL) and the product ofstep 1G (40 mg, 77 μmol) added, along with HBTU (33.2 mg, 87 μmol) anddiisopropylethylamine (100 μL, 560 μmol). This was stirred for 18 hr.The reaction was concentrated, and the residue dissolved in ethylacetate. The organics were washed with water, 10% potassium hydrogensulfate, water, and brine, and then concentrated. The residual oil waspurified by flash chromatography on silica (EtOAc/2-PrOH, 1%->10%) andthe product fractions combined and evaporated to yield the product (36mg, 53%) as a white solid. LRMS (ES): 983.6 [M+H]⁺, 492.5 [M+2H]⁺²; HRMS(ESI): Calculated for C₅₁H₆₇N₈O_(12—983.4878,) found—983.4860; ¹HNMR(600.1300 MHz, CDCl₃) 7.63 (b, 2H), 7.45 (b, 1H) 7.22-7.41 (m, 11H),6.90 (b, 1H), 6.54 (d, 1H), 5.99 (b, 1H) 5.39 (d, J=16.6 Hz, 1H), 5.12(m, 3H), 4.78-4.98 (m, 2H), 4.51 (b, 1H), 4.40 (b, 1H),4.25 (b, 1H),3.87 (d, J=16.6 Hz 1H), 3.76 (s, 3H), 3.66 (b, 1H), 3.45 (b, 1H), 3.19(s, 3H), 3.17 (m, 1H), 3.03 (m, 2H), 2.69 (dd, 1H), 2.25-2.45 (m, 4H)2.05-2.16 (m, 2H), 1.96 (m, 2H), 1.71 (m, 2H), 1.46 (s, 9 H), 1.44 (s,9H).

[0420] Step 1I: Synthesis of tert-butyl(S,S,S)-4-amino-4-(N-(3-(3,6-diaza-5-((methoxycarbonyl)methyl)-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)butanoate acetate salt

[0421] The product of Step 1H (33 mg, 33 μmol) was hydrogenated with 10%palladium on carbon (15 mg) in methanol (6 mL) with acetic acid (0.1 mL)on a Parr shaker at 40 psi for 1.5 hr. The solution was filtered onCelite, rinsed with methanol and concentrated. The residue was dissolvedin 20 mL 1:1 acetonitrile/water, frozen, and lyophilized to afford theproduct as a white powder (21 mg, 75%). LRMS (ES): 849.5 [M+H]⁺, 425.5[M+2H]⁺²;

[0422] Step J: Synthesis of tert-butyl(S,S,S)-4-(N-(1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,5,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoate trifluoroacetate

[0423] The product of step 1I (20 mg, 16.8 μmol) was dissolved in DMF (1mL) along with DOTA(OtBu)3-OH (26 mg, 25 μmol), HBTU (20 mg, 53 μmol),diisopropylethylamine (29.1 mg, 225 μmol) and HOBT hydrate (2.5 mg, 18μmol). This was stirred for 18 hr under nitrogen, concentrated undervacuum, and purified by preparative HPLC (Vydac C-18, 2.5 cm×15 cm, 0.1%TFA/acetonitrile gradient). The product fractions were pooled andlyophilized to afford 17.5 mg of product as a white powder. LRMS (ES)589.5, 617.8, 646.1, 674.5 [(M-ntBu)+2H]+2, 702.8 [M+2H]+2, 1403.9[M+H]⁺

[0424] Step 1K: Synthesis of(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(14,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoic acid

[0425] The product of I (16 mg, 7.67 μmol (as 6TFA salt)) was dissolvedin THF/MeOH (1:1, 1 mL) and lithium hydroxide added (26 μL of a 3Msolution in water). The reaction was stirred for 2 hr, concentrated, andtreated with trifluoroacetic acid (0.8 mL) and triethylsilane (0.2 mL)under nitrogen. The solution was stirred for 21 hr, concentrated undervacuum, and purified by preparative HPLC (Vydac C-18, 21.5 mm×15 cm,0.1% TFA/acetonitrile gradient). The product fractions were pooled andlyophilized to afford the product (6.5 mg, 55%) as a white powder. LRMS(ES): 370.9 [M+3H]+3, 555.6 [M+2H]+2, 1109.5 [M+H]+; HRMS: Calculatedfor C₅₀H₆₉O₁₇N₁₂: 1109.4904, found: 1109.4890.

Example 2 Preparation of(S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid trifluoroacetate salt

[0426]

[0427] Step 2A: Synthesis of tert-butyl 3-(((6-((tert-butoxy)carbonylamino)hexyl)amino)methyl)-4-fluorobenzoate

[0428] This was prepared in the same fashion as Example 1A fromtert-butyl-4-fluoro-3(alpha-bromomethyl)benzoate (5.4 g., 18 mmol) and6-tert-butoxycarbonylamino-1-hexylamine hydrochloride (5.0 g., 19.8mmol), affording 3.1 g (41%) of product as a yellow oil. LRMS: 425.2[M+H]⁺; ¹HNMR (270 MHz, DMSO-d6): 7.95 (dd, 1H), 7.87 (dd, 1H), 7.04 (t,1H), 4.50 (bs, 1H), 3.83 (s, 2H), 3.07 (q, 2H), 2.59 (t, 2H), 1.57 (s,9H), 1.42 (s, 9H), 1.60-1.20 (m, 8H);

[0429] Step 2B: Synthesis of methyl (S)-3-N-(6-((tert-butoxyl)carbonylamino)hexyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate

[0430] This was prepared as in Example 1B, starting with 3.06 g ofamine, affording 4.4 g(88%) of the product as a viscous oil. LRMS: 688.4[M+H]⁺; ¹HNMR (270 MHz, DMSO-d6): Mixture of amide rotamers, 7.85 (m,2H), 7.80 (d, 1H) , 7.4-7.2 (m, 6H), 6.73 (br t, 1H), 5.10-4.40 (m, 4H),3.56, 3.53 (2s, 3H), 3.35 (m, 2H), 3.00-2.55 (m, 4H), 1.51 (s, 9H), 1.35(s, 9H), 1.70-1.10 (m, 8H);

[0431] Step 2C: Synthesis of methyl(S)-3-amino-3-(N-(6-((tert-butoxy)carbonylamino)hexyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)propanoate

[0432] This step was done in the same fashion as Example 1C, startingwith 2.3 g of CbZ protected compound, affording 1.71 g (92%) of theamine as a pale yellow oil. LRMS: 554.3 [M+H]⁺; ¹HNMR (270 MHz, DMSO-d6)mixture of amide rotamers: 7.90-7.70 (m, 2H), 7.29 (m, 1H), 6.75 (br,1H), 4.80 (q, 1H), 4.54 (s, 2H), 4.10 (q, 1H), 3.89 (2t, 1H), 3.53 (2s,3H) 2.87 (m, 2H), 2.55 (m, 2H), 1.90 (bs, 1H), 1.52 (s, 9H), 1.35 (s,9H), 1.70-1.10 (m, 8H);

[0433] Step 2D: Synthesis of methyl (S)-2-(2,5-diaza-9-((tert-butyl)oxycarbonyl)-5-(6-((tert-butoxy)carbonylamino)hexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0434] This step was done in the same fashion as Example 1D, startingwith 1.66 g of amine, affording 706 mg (44%) of the benzodiazepine as apale yellow foam. LRMS: 534.3 [M+H]⁺; ¹HNMR (270 MHz, DMSO-d6) mixtureof amide rotamers: 7.55 (d, 1H), 7.50 (dd, 1H), 6.70 (br t, 1H), 6.55(br, 1H), 6.54 (d, 1H), 5.40 (d, 1H), 5.14 (m, 1H), 3.99 (d, 1H), 3.59(s, 3H) 2.78 (m, 2H), 2.65 (q, 2H), 1.49 (s, 9H), 1.35 (s, 9H),1.30-1.00 (m, 8H);

[0435] Step 2E: Synthesis of (S)-2,5-diaza-5-(6-((tert-butoxy)carbonylamino)hexyl)-3-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-9-carboxylicacid

[0436] This step was done in the same fashion as Example 1E, startingwith 301 mg of ester, affording the crude product (394 mg) as a yellowfoam, which was used directly in the next step without purification.LRMS: 478.2 [M+H]⁺.

[0437] Step 2F: Synthesis of methyl(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-((tert-butoxy)carbonylamino)hexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0438] The reaction was carried out as in Example 1F, obtaining 306 mgof crude solid, which was further purified by flash chromatography toafford the desired product (164 mg, 47% from Step D) as a pale yellowsolid. LRMS: 621.3 [M+H]⁺; ¹HNMR (270 MHz, DMSO-d6): 12.40 (br, 1H),7.53 (bs, 2H), 7.20 (m, 4H), 6.71 (br, 1H), 6.52 (d, 1H), 6.23 (bd, 1H),5.40 (d, 1H), 5.10, (m, 1H), 4.76 (s, 2H), 3.85 (bd, 1H), 3.59 (s, 3H),3.04 (s, 3H), 2.90-2.55 (m, 2H), 1.35 (s, 9H), 1.40-1.20 (m, 8H).

[0439] Step 2G: Synthesis of(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-((tert-butoxy)carbonylamino)hexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid

[0440] The product of step F (152 mg, 245 μmol) was stirred with lithiumhydroxide (21 mg, 500 μmol) in THF/H2O (3 mL/2 mL) for 22 hr. THF wasremoved under vacuum, the residue diluted with water and acidified withsolid citric acid. The precipitated solid and solution was extractedwith dichloromethane, washed with brine, dried (Na₂SO₄), andconcentrated to afford the acid product (120 mg, 81%) as a pale yellowpowder, which was not purified further. LRMS: 607.2 [M+H]⁺.

[0441] Step 2H: Synthesis of(S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid trifluoroacetate

[0442] The product of step G (87 mg, 143 μmol) was dissolved in CH₂Cl₂(4 mL) and trifluoroacetic acid (2 mL) added with stirring undernitrogen. The solution was stirred for one hour, concentrated undervacuum, and the residue redissolved in dry DMF (2.5 mL). To this wasadded sodium 2-[[[5-[[(2,5-dioxo-1-pyrollidinyl)oxy]carbonyl]-2-pyridinyl] hydrazono]methyl]-benzenesulfonate (75 mg,170 μmol) and diisopropylethylamine (500 μL, 2.87 mmol)with stirringunder nitrogen. The reaction was stirred overnight, concentrated, andthe residue purified by preparative HPLC (Vydac C-18, 2.5 cm×15 cm, 0.1%TFA/acetonitrile gradient). The product fractions were combined andlyophilized to afford the product as a pale yellow powder (47.3 mg,35%). LRMS (ES): 810.3 [M+H]⁺. ¹HNMR (600.1300 MHz, DMSO-d6): 12.40 (b,2H), 9.24 (bs, 1H), 8.59 (bs, 1H), 8.50 (s, 1H), 8.24 (bs, 1H), 8.20(bs, 1H), 7.80 (d, 3H), 7.53 (m, 2H), 7.41 (m, 2H), 7.20 (m, 3H), 6.57(d, 1H), 6.32 (bs, 1H), 5.40 (d, 1H, J=16.4 Hz), 5.10 (m, 1H), 4.76 (s,2H), 3.85 (d, 1H, J=16.4 Hz), 3.55 (m, 2H), 3.21 (m, 2H), 3.04 (s, 3H),2.79 (dd, 1 H, J=16.5 Hz, 9 Hz), 2.55 (dd, 1H, J=16.5 Hz, 5 Hz), 1.60(m, 2H), 1.51 (m, 2H), 1.26 (m, 2H), 1.19 (m, 2H).

Example 3 Synthesis of(S)-2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-N-benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid trifluoroacetate

[0443]

[0444] Step 3A: Synthesis of N-(6-((benzimidazol-2-ylmethyl)amino)hexyl)(phenylmethoxy)formamide dihydrochloride

[0445] Both α-bromomethyl-(N-tert-butoxycarbonyl)benzimidazole (3.42 g,11 mmol, prepared according to WO96/00730) andN-(mono-benzyloxycarbonyl)-hexanediamine (4.58 g, 16 mmol, preparedaccording to Bioconj. Chem., 1997, 8, 611) were dissolved in THF (100mL), along with diisopropylethylamine (8 mL, 45.9 mmol) and water (3mL). The mixture was stirred for 20 hr, concentrated, and the residuepartitioned between 1N NaOH and dichloromethane. The aqueous wasreextracted and concentrated to afford a yellow semi-solid product whichwas dissolved in ether/dichloromethane (2:1, 300 mL) and treated with 4NHCl in dioxane (40 mL, 160 mmol) with stirring at room temperature for18 hr. The resulting solids were filtered, dissolved in a minimum amountof 10% sodium carbonate, extracted into dichloromethane and concentratedto an oil. This was purified by flash chromatography on silica (9:1EtOAc/EtOH, 0.1% NH₄OH) and the product fractions concentrated,dissolved in ether, and treated with 4N HCl/dioxane. The resultingsolids were filtered and washed with ether to afford 745 mg of a whitepowder. LRMS: 381.3 [M+H]⁺; ¹HNMR (270 MHz, DMSO-d6): 10.04 (b, 2H),7.78 (m, 2H), 7.44 (m, 2H), 7.34 (m, 6H) 6.76 (b, 2H), 4.99 (s, 2H),4.60 (s, 2H), 3.10 (m, 2H), 2.99 (m, 2H), 1.67 (m, 2H), 1.41 (m, 2H),1.29 (m, 4H)

[0446] Step 3B: Synthesis of methyl(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-(6-((phenylmethoxy)carbonylamino)hexyl)carbamoyl)-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0447] The product of Step 3A (300 mg, 0.66 mmol), methyl(S)-(-)-7-carboxy-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetate(172 mg, 0.55 mmol, prepared according to PCT/US95/00248, WO 95/18619),HOBT (89 mg, 0.66 mmol), and diisopropylethylamine (380 μL, 2.18 mmol)were dissolved in dry DMF (5 mL) in dry glassware under nitrogen. EDC(89 mg, 0.66 mmol) was added in one portion and the reaction stirred 20hr. The solution was concentrated, partitioned between water and ethylacetate, and the aqueous layer extracted with two additional portions ofethyl acetate. The combined organics were washed with water and brine,and concentrated. The crude oil was purified by flash chromatography onsilica gel (EtOAc, 0.5% EtOH). The product fractions were combined andconcentrated to yield 145 mg (40%) of product as a light brown solid.LRMS (ES): 655.3 [M+H]⁺; ¹HNMR (600.1343 MHz, DMSO-d6): 12.38 (b, 1H),7.51 (m, 2H), 7.30 (m, 6H), 7.14 (m, 4H), 6.51 (d, 1H), 6.16 (d, 1H),5.42 (d, 1H, J=16 Hz), 5.08 (m, 1H), 4.96 (s, 2H), 4.73 (s, 2H), 3.88(d, 1H, J=16 Hz), 3.57 (s, 3H), 3.33 (m, 2H), 2.89 (m, 2H), 2.85 (s,3H), 2.78 (dd, 1 H, J=16.5 Hz, 9 Hz), 2.61 (dd, J=16.5 Hz, 5 Hz), 1.52(m, 2H), 1.30 (m, 2H), 1.15 (m, 4H); ¹³C NMR (600.1343 MHz, DMSO-d6):170.9, 169.1, 165.6, 156.0, 151.3, 147.4, 137.3, 129.3, 128.3, 127.8,127.7, 127.3, 123.0, 118.1, 114.9, 65.0, 59.7, 51.6, 51.3, 50.1, 50.0,37.4, 35.0, 29.5, 29.2, 26.6, 20.7, 14.1

[0448] Step 3C: Synthesis of methyl(S)-2-(9-(N-(6-aminohexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-2,5-diaza-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0449] The product of 3B (140 mg, 214 μmol) was dissolved in methanol (6mL) with 10% palladium on carbon (30 mg). The slurry was hydrogenated atone atmosphere pressure for 5.5 hr, filtered through Celite® andconcentrated to yield the product (100 mg, 90%) as a clear oil which wasnot further purified, but taken directly into the next step. LRMS (ES)521.4 [M+H]⁺, 275.3, 261.3, 245.2, 231.3.

[0450] Step 3D: Synthesis of(S)-2-(9-(N-(6-aminohexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-2,5-diaza-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

[0451] The product of Step 3C (100 mg, 192 μmol) was dissolved inmethanol/tetrahydrofuran (2:1, 1 mL) and lithium hydroxide hydrate (23mg, 550 μmol) dissolved in 0.5 mL water was added. The reaction wasstirred for 4 hr, neutralized with 10% potassium hydrogen sulfatesolution, and concentrated. The solids were dissolved in methanol,filtered, and the filtrate concentrated to an oil, which was dissolvedin water/acetonitrile and lyophilized to afford 93 mg (96%) of theproduct as a white solid. LRMS (ES): 507.3 [M+H]⁺, 459.4, 254.4[M+2H]⁺²; ¹HNMR (600.1300 MHz, DMSO-d6): 12.35 (b, 1H), 10.49 (b, 3H),7.59 (m, 2H), 7.53 (m, 2H), 7.16 (bs, 4H), 6.53 (d, 1H, J=7.4 Hz), 6.18(s, 1H), 5.44 (d, 1H, J=16.4 Hz), 5.08 (m, 1H), 4.76 (s, 2H), 3.80 (bd,1H, J=12 Hz), 3.38 (m, 2H), 2.88 (s, 3H), 2.78 (dd, 1 H, J=16.7 Hz, 9Hz), 2.71 (m, 2H), 2.61 (dd, 1H, J=16.7 Hz, 5 Hz), 1.55 (m, 2H) , 1.47(m, 2H) , 1.18 (m, 2H) , 1.03 (m, 2H)

[0452] Step 3E: Synthesis of2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid trifluoroacetate

[0453] The product of Step D (80 mg, 160 μmol) was dissolved in drydimethylformamide, along with sodium 2-[[[5-[[(2,5-dioxo-1-pyrolidinyl)oxy]carbonyl]-2-pyridinyl] hydrazono]methyl]-benzenesulfonate (88 mg,250 μmol) and diisopropylethylamine (280 μL, 1.6 mmol) with stirringunder nitrogen. The reaction was stirred overnight, concentrated, andthe residue purified by preparative HPLC (Vydac C-18, 21.5 mm×25 cm,0.1% TFA/acetonitrile gradient). The product fractions were combined andlyophilized to afford the product as a white solid (24 mg, 18%). LRMS(ES): 810.3 [M+H]⁺, 4764.3, 399.3; HRMS (ESI): Calculated forC₄₀H₄₄N₉O₈S (M+H)—810.3033, found—810.3052. ¹HNMR (600.1300 MHz,DMSO-d6): 12.40 (b, 2H), 9.24 (bs, 1H), 8.59 (bs, 1H), 8.50 (s, 1H),8.24 (bs, 1H), 8.20 (bs, 1H), 7.80 (d, 3H), 7.53 (m, 2H), 7.41 (m, 2H),7.20 (m, 3H), 6.57 (d, 1H), 6.32 (bs, 1H), 5.47 (d, 1H, J=16.4 Hz), 5.08(m, 1H), 4.98 (s, 2H), 3.83 (d, 1H, J=16.4 Hz), 3.50 (m, 2H), 3.21 (m,2H), 2.89 (s, 3H), 2.75 (dd, 1 H, J=16.7 Hz, 9 Hz), 2.53 (dd, 1H, J=16.7Hz, 5 Hz), 1.65 (m, 2H) , 1.48 (m, 2H) , 1.26 (m, 2H), 1.19 (m, 2H)

Example 4 Preparation of(S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic acid

[0454]

[0455] Step 4A. Synthesis of(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

[0456] The product of Step 2E (350 mg, 564 μmol) was dissolved inmethanol/tetrahydrofuran (2:1, 8 mL) with stirring. Lithium hydroxidehydrate (95 mg, 2.25 mmol) was dissolved in water (5 mL) and added tothis solution. It was stirred for two hours, neutralized with 10%potassium hydrogen sulfate and concentrated to a gummy solid. This wasadded to a solution of trifluoroacetic acid in dichloromethane (4 mL/6mL) and stirred for two hours. The solids were filtered off, and thefiltrate concentrated to afford an oil, which was redissolved inwater/acetonitrile and lyophilized to a white powder which was notfurther purified. LRMS (ES): 507.4 [M+H]⁺, 254.4 [M+2H]⁺².

[0457] Step 4B. Synthesis of(S,S)-2-(2,5-diaza-(9-(N-benzimidazol-2-ylmethyl))-5-(6-(4-(N-(6-(3,6-diaza-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-2-((tert-butoxy)carbonylamino)butanoylamino)hexyl)-4-oxobicyclo[5.4.0]undeca-1(11),7(8),9-trien-3-yl)aceticacid

[0458] The product of 4A (31 mg, 36.5 μmol) was dissolved in drydimethylformamide (1.5 mL), along with diisopropylethylamine (51 μL, 300μmol). To this was addedbis-(N-hydroxysuccinimide)-N-(tert-butoxycarbonyl)-glutamate (7.7 mg,17.5 μmol) with stirring. The solution was allowed to stir for threehours, when it was concentrated and purified by preparative HPLC (VydacC-18, 21.5 mm×25 cm, 0.1% TFA/acetonitrile gradient). The productfractions were combined and lyophilized to afford the product as a whitesolid (12 mg, 33%). LRMS (ES): 1224.7 [M+H]⁺, 613.1 [M+2H]⁺², 409.3[M+3H]⁺³. HRMS (ESI): Calculated for C₆₄H₈₂N₁₃O₁₂—1224.6206,found—1224.619.

[0459] Step 4C. Synthesis of(S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic acid

[0460] The product of 4B (10 mg, 5.5 μmol of 4TFA salt) was dissolved indichloromethane:triflouroacetic acid (1.5 mL/0.5 mL) under nitrogen. Itwas stirred 20 minutes and concentrated to an oil, which was resuspendedin toluene and reconcentrated to remove residual TFA. The residue wastreated as in step 3E to afford 2.5 mg (31%) of the product as a whitelyophilized solid. LRMS (ES): 1428.2 [M+H]⁺, 714.5 [M+2H]⁺², 477.3[M+3H]⁺³. HRMS (ESI): Calculated for C₇₂H₈₃N₁₆O₁₄S—1427.5995,found—1427.601.

Example 5 Preparation of(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino) butanoic acid

[0461]

[0462] Step 5A. Synthesis ofbenzyl((1-(triphenylmethyl)imidazol-2-yl)methyl)amine

[0463] N-tritylimidazole-2-carboxaldehyde (338 mg, 1 mmol, preparedaccording to K. L. Kirk; J.Org.Chem., 1978, 43, 4381) was dissolved indry toluene (7 mL) and anhydrous magnesium sulfate (602 mg, 5 mmol)added with stirring under nitrogen. Benzylamine (131 μL, 1.2 mmol) wasadded and the solution stirred for 3.5 hr. The solids were filteredunder nitrogen and the reaction concentrated. The residue is redissolvedin 1,2-dichloroethane (25 mL) and cooled to 0° C. Sodiumtriacetoxyborohydride (1.06 g, 5 mmol) was added slowly. The solutionwas allowed to warm to room temperature over 2.5 hours. The reactionmixture was added to water/ethyl acetate and the layers separated. Theaqueous layer was extracted with two portions of ethyl acetate and thecombined organic layers washed with sat. bicarbonate, water, and brine.The solution was concentrated to an oil and purified by flashchromatography on silica gel (99:1 EtOAc/EtOH with 0.1% triethylamine)to afford 330 mg (77%) of product as an oil which solidified onstanding. LRMS (ES): 430.4 [M+H]⁺, 243.2; ¹HNMR (600.1328 MHz, DMSO-d6):7.37 (m, 11H), 7.04 (m, 9 H), 6.92 (d, 1H), 6.64 (d, 1H), 3.34 (s, 2H),2.77 (2H).

[0464] Step 5B. Synthesis of methyl(S)-2-(2,5-diaza-5-(3-((tert-butoxy)carbonylamino)propyl)-4-oxo-9-(N-benzyl-N-((2-(triphenylmethyl)imidazol-2-yl)methyl)carbamoyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3yl)acetate

[0465] The product of step 5E (150 mg, 0.345 mmol) was treated in thesame manner as step 1F, affording the product (250 mg, 85%) as a thickoil. LRMS (ES): 847.5 [M+H]⁺, 430.5, 243.2; ¹HNMR (600.1330 MHz, CDCl₃)This sample gave broad peaks with little fine splitting, even whenrefiltered, and was qualitatively similar to 1E for the benzodiazepinenucleus.

[0466] Step 5C. Synthesis of methyl(S)-2-(5-(3-aminopropyl)-2,5-diaza-9-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0467] The product of step 5B (220 mg, 0.26 mmol) was added to neattrifluoroacetic acid (4 mL) containing triethylsilane (1 mL) undernitrogen and stirred for 1.5 hr. The solution was concentrated andresidual acid removed by reconcentration with toluene. This product wasnot purified, but was used directly in the following step. LRMS (ES):505.4 [M+H]⁺, 253.4.

[0468] Step 5D. Synthesis of tert-butyl(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)butanoate

[0469] A portion of the product of step 5C (65 mg, 130 μmol) was reactedwith step 1G as in Step 1H to afford the product (64 mg, 49% from 5B) asan oil. LRMS (ES): 1009.7 [M+H]⁺, 505.6 [M+2H]⁺²,; HRMS (ESI):Calculated for C₅₃H₆₉N₈O₁₂₋—1009.5035, found—1009.502; ¹HNMR (600.1330MHz, CDCl₃) 7.47 (b, 1H), 7.22-7.41 (m, 14H), 6.99 (s, 2H), 6.93 (b,1H), 6.44 (d, 1H), 5.98 (b, 1H) 5.32 (d, 1H), 5.13 (d, 1H), 5.05 (m, 2H)4.68 (m, 3H), 4.48 (b, 1H), 4.36 (b, 1H),4.24 (b, 1H), 3.71 (s, 3H),3.68 (m, 1H), 3.60 (b, 1H), 3.38 (b, 1H), 3.11 (b, 1H), 2.97 dd, 1H),2.94 (m, 1H), 2.65 (dd, 1H), 2.25-2.45 (m, 4H) 1.88-2.16 (m, 4H), 1.65(m, 2H), 1.45 (s, 9 H), 1.41 (s, 9H).

[0470] Step 5E: Synthesis of tert-butyl(S,S,S)-4-amino-4-(N-(1-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)butanoate

[0471] The product of 5D (58 mg, 57 μmol) was hydrogenated according tothe procedure of step 1I, to yield the product (44 mg, 88%) as a whitesolid, which was not further purified but was lyophilized in 0.1%aqueous trifluoroacetic acid/acetonitrile (1:1) and used as thetrifluoroacetate salt in the next step. LRMS (ES): 875.6 [M+H]⁺, 438.5[M+2H]⁺²,;

[0472] Step 5F: Synthesis of tert-butyl(S,S,S)-4-(N-(1-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoate

[0473] The product of 5E (24.4 mg, 20 μmol) was reacted with DOTAtri-tert-butyl ester as in step 1J, to afford the product (19.6 mg, 55%)as a trifluoroacetate salt after lyophilization.

[0474] LRMS (ES): 1430.0 [M+H]⁺, 715.7 [M+2H]⁺², 477.8 [M+3H]⁺³;HRMS(ESI): Calculated for C₇₃H₁₁₃N₁₂O_(17—-)1429.8347, found13 1429.838;

[0475] Step 5G: Synthesis of(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino)butanoic acid

[0476] The product of 5F (13 mg, 7.4 μmol) was deprotected and purifiedas in step 1K, to afford the product (6.5 mg, 55%) as a trifluoroacetatesalt after lyophilization. LRMS (ES): 1135.6 [M+H]⁺, 568.5 [M+2H]⁺²,379.6 [M+3H]⁺³; HRMS(ESI): Calculated for C₅₂H₇₁N₁₂O_(17—)1135.5060,found—1135.503;

Example 6 Preparation of(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoic acid

[0477]

[0478] Step 6A: Synthesis of tert-butyl(S,S)-3-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate

[0479] The product of step 5D (65 mg, 130 μmol) was reacted withN-(carbobenzyloxy)-β-(tert-butyl)-□-(N-hydroxysuccinimidyl) aspartate(66 mg, 156 μmol) and diisopropylethylamine (181 μL, 1.04 mmol) indimethylformamide (1.5 mL) with stirring at room temperature undernitrogen for 20 hr. The reaction was concentrated, and the residuedissolved in ethyl acetate. The organics were washed with water, 10%potassium hydrogen sulfate, water, and brine, and then concentrated. Theresidual oil was purified by flash chromatography on silica (EtOAc/MeOH,1%->10%) and the product fractions combined and evaporated to yield theproduct (76 mg, 73%) as an oil. LRMS (ES): 810.5 [M+H]⁺, 378.0; HRMS(ESI): Calculated for C₄₃H₅₂N₇O_(9—)810.3826, found—810.3819; ¹HNMR(600.1323 MHz, CDCl₃) 7.25-7.38 (m, 12H), 7.18 (m, 2H), 7.07 (b, 1H),6.99 (s, 2H), 6.39 (d, 1H), 6.18 (b, 1H) 5.30 (d, J=16.2 Hz, 1H), 5.09(m, 2H), 5.04 (m, 1H) 4.67 (m, 4H), 4.50 (b, 1H), 4.36 (b, 1H), 3.69 (s,3H), 3.62 (d, J=18.6 Hz, 1H), 3.45 (b, 1H), 3.14 (m, 1H), 2.94 (dd, 1H),2.86 (m, 2H), 2.62 (m, 2H), 1.60 (m, 2H), 1.39 (s, 9 H).

[0480] Step 6B: Synthesis of tert-butyl(S,S)-3-amino-3-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)propanoate

[0481] The product of 6A (70 mg, 86 μmol) was hydrogenated according tothe procedure of step 1I, to yield the product (55 mg, 95%) as a whitesolid, which was not further purified but was lyophilized in 0.1%aqueous trifluoroacetic acid/acetonitrile (1:1) and used as thetrifluoroacetate salt in the next step. LRMS (ES): 676.5 [M+H]⁺, 339.0[M+2H]⁺², 310.9.

[0482] Step 6C: Synthesis of tert-butyl(S,S)-3-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)propanoate

[0483] The product of 6B (22.4 mg, 22 μmol) was reacted with DOTAtri-tert-butyl ester and purified as in step 1J, to afford the product(16.6 mg, 44%) as a trifluoroacetate salt after lyophilization. LRMS(ES): 1230.9 [M+H]⁺, 616.2 [M+2H]⁺², 411.3 [M+3H]⁺³; HRMS(ESI):Calculated for C₆₃H₉₆N₁₁O_(14—)1230.7138, found—1230.715;

[0484] Step 6D: Synthesis of(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoic acid

[0485] The product of 5F (14 mg, 8.3 μmol) was deprotected and purifiedas in step 1K, to afford the product (4.6 mg, 47%) as a trifluoroacetatesalt after lyophilization. LRMS (ES): 992.6 [M+H]⁺, 497.0 [M+2H]⁺²,331.8 [M+3H]⁺³; HRMS(ESI): Calculated for C₄₆H₆₂N₁₁O_(14—-)992.4478,found—992.4457;

Example 7 Synthesis of(S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4,4-dihydroxypentyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoic acid

[0486]

[0487] Step 7A: Synthesis of tert-butyl(S,S,S,S,S,S)4-(N-(1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-(N-(1-(N-(1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl) carbamoyl)-2-((phenylmethoxy)carbonylamino)butanoylamino) butanoate

[0488] The product of step 1I (65 mg, 54.6 μmol) is dissolved in DMF (1mL) along with HBTU (25 mg, 65 μmol), N-carbobenzyloxy-L-glutamic acid(7.3 mg, 26 μmol), HOBT (7 mg, 52 μmol), and diisopropylethylamine (40μL, 225 μmol) under nitrogen. After stirring for 2 hrs, the reaction isconcentrated and purified by preparative HPLC (0.1% TFA/acetonitrilegradient, Zorbax C8, 21.5 mm×25 cm). The product may be obtained as thetrifluoroacetate salt after lyophilization.

[0489] Step 7B: Synthesis of tert-butyl(S,S,S,S,S,S)-4-(2-amino-4-(N-(1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)butanoylamino)-4-(N-(1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)butanoate

[0490] The product of step 7A is hydrogenated and isolated as in step1I. This material is not further purified, but used directly in thefollowing step.

[0491] Step 7C: Synthesis of tert-butyl(S,S,S,S,S,S,S,S)-4-(N-(1,3-bis(N-(3-((tert-butyl)oxycarbonyl)-1-(N-3-((tert-butyl)oxycarbonyl)-1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-4-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)butanoate

[0492] The product of step 7B is reacted as in step 5D to afford theproduct, which is purified by preparative HPLC.

[0493] Step 7D: Synthesis of tert-butyl(S,S,S,S,S,S,S,S)-4-amino-4-(N-(1-(N-(1,3-bis(N-(3-((tert-butyl)oxycarbonyl)-1-(N-3-((tert-butyl)oxycarbonyl)-1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)butanoate

[0494] The product of step 7C is hydrogenated as in step 1I to affordthe amine, which is not further purified but used directly in the nextstep.

[0495] Step 7E: Synthesis of tert-butyl(S,S,S,S,S,S,S,S)-4-(N-(1-(N-(1,3-bis(N-(3-((tert-butyl)oxycarbonyl)-1-(N-3-((tert-butyl)oxycarbonyl)-1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl))propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoate

[0496] The product of step 7D is reacted with DOTA(OtBu)3-OH as in step1J to afford the product as a solid after preparative HPLC purificationand lyophilization. Alternatively, the product of 7B is reacted with theproduct of 7I in the presence of HBTU, HOBT, and diisopropylethylaminein dry dimethylformamide for 2 hours, after which the reaction isconcentrated and the residue purified by preparative HPLC to afford theproduct as a solid after lyophilization.

[0497] Step 7F: Synthesis of(S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4,4-dihydroxypentyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoic acid

[0498] The product of step 7D is deprotected as in step 1K to afford theproduct as a solid after preparative HPLC purification andlyophilization.

[0499] Step 7G: Synthesis of tert-butyl (S,S)-3,3-dimethyl-3-silabutyl2-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)pentane-1,5-dioate

[0500] The product of step 1G (1.25 g, 2.4 mmol) was reacted with2-trimethylsilylethanol (296 mg, 2.5 mmol) in the presence of ethyl[3-(N,N-dimethylaminopropyl]-carbodiimide hydrochloride (480 mg, 2.5mmol) and dimethylaminopyridine (250 mg, 1.2 mmol) in dimethylformamide(10 mL) at 0° C. The reaction was allowed to warm slowly to roomtemperature and stirred overnight. It was concentrated and the residuepartitioned between ethyl acetate and water. The aqueous layer wasextracted twice with ethyl acetate, and the combined organics washedwith water, 10% potassium hydrogen sulfate, and brine, and concentrated.The residue was purified by flash chromatography (ethyl acetate/hexane)to afford the product as an oil (1.1 g, 73%). LRMS (ES): 623.5 [M+H]⁺.

[0501] Step 7H: Synthesis of tert-butyl (S,S)-3,3-dimethyl-3-silabutyl2-(2-amino-4-((tert-butyl)oxycarbonyl)butanoylamino)pentane-1,5-dioate

[0502] The product of step 7G (1.09 g) was dissolved in 2-propanol (75mL) with 10% palladium on carbon (300 mg) and hydrogenated on a Parrshaker at 45 psi for one hour. The reaction mixture was filtered on abed of Celite, washed with 2-propanol, and concentrated to yield theproduct (803 mg, 94%) as a clear oil. LRMS (ES): 489.5 [M+H]⁺, 977.7[2M+H]⁺. ¹HNMR (600.1343 MHz, CDCl₃): 7.78 (m, 1H), 4.53 (m, 1H), 4.22(m, 2H), 3.53 (m, 1H), 1.80-2.41 (m, 10H), 1.43 (s, 18H), 1.01 (m, 2H),0.02 (s, 9H).

[0503] Step 7I: Synthesis of tert-butyl (S,S)-3,3-dimethyl-3-silabutyl2-(4-((tert-butyl)oxycarbonyl)-2-(2-bromoacetylamino)butanoylamino)pentane-1,5-dioate

[0504] The product of step 7H (397 mg, 0.813 mmol) was dissolved in drytetrahydrofuran (5 mL) with diisopropylethylamine (180 μL, 1.05 mmol)and cooled to −10° C. under nitrogen. Bromoacetyl bromide (85 μL, 0.98mmol), dissolved in 10 mL tetrahydrofuran, was added dropwise to thecold solution, keeping T≦−5° C. The reaction was stirred in the cold for1.5 hr, and 25 μL methanol added. The solids were filtered and rinsedand the combined filtrate concentrated to a brown oil, which waspurified by flash chromatography (dichloromethane/ethyl acetate) toafford the product (388 mg, 78%) as a light tan oil. LRMS (ES):609.3/611.3 [M+H]⁺, 631.3/633.3 [M+Na]⁺, 185.3, 144.2. ¹HNMR (600.1343MHz, CDCl₃): 7.32 (m, 1H), 7.09 (m, 1H), 4.50 (m, 2H), 4.21 (m, 2H),3.87 (m, 2H), 2.31 (m, 2H), 2.13 (m, 2H), 1.99 (m, 2H), 1.97 (m, 2H),1.45 (s, 9H), 1.43 (s, 9H), 1.01 (m, 2H), 0.04 (s, 9H).

[0505] Step 7J: Synthesis of(S,S)-4-((tert-butyl)oxycarbonyl)-2-(4-((tert-butyl)oxycarbonyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoylamino)butanoic acid

[0506] The product of step 7H (214 mg, 0.416 mmol) was dissolved indimethylformamide (3 mL) and added to a solution of triethylamine (250μL) and DO3A tri-tert-butyl ester in dimethylformamide (3mL). Thereaction was stirred for 4 days at room temperature, concentrated, andthe residue dissolved in ethyl acetate. This was washed with water andbrine, dried, and concentrated to an oil which was not further purifiedbut reacted directly with tetra-butylammonium fluoride (1.0M intetrahydrofuran, 1.25 mL) in tetrahydrofuran (2.5 mL). After stirringfor 2 hours, the reaction was treated with ether (50 mL) and water (50mL) and the layers separated. The aqueous layer was extracted with threeportions of ethyl acetate, and the combined organic layers concentratedto an oil. This was purified by preparative HPLC (0.1% trifluoroaceticacid/acetonitrile, Zorbax C-8, 21.5 mm×25 cm) and the product fractionslyophilized to afford 127 mg (32% for two steps) of the product as awhite solid. LRMS (ES): 943.3 [M+H]⁺, 887.2, 831.2, 775.5, 719.3, 663.2(loss of 1-5 tert-butyl) 444.3, 416.2, 388.3, 360.1, 332.1 [M-(1-5 tertbutyl)+2H]⁺². ¹HNMR (600.1343 MHz, CDCl₃): 9.05 (b, 1H), 8.2 (b, 4H)7.36 (b, 1H), 4.34 (m, 2H), 2.77-4.23 (very broad humps, 24H), 2.31 (m,4H), 2.13 (m, 2H), 1.93 (m, 2H), 1.47 (d, 18H), 1.43 (m, 27H).

Example 8 Synthesis of(S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-4-(2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)-4-carboxybutanoylamino)-4-carboxybutanoylamino)butanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)butanoic acid

[0507]

[0508] Step 8A: Synthesis of ditert-butyl (S,S)-2-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)pentane-1,5-dioate

[0509] Gamma-tert-butyl-N-carbobenzyloxyglutamic acidN-hydroxy-succinimide ester is dissolved in DMF withdiisopropylethylamine. Bis(tert-butyl)glutamate hydrochloride is addedand the reaction stirred for one hour. The reaction is concentrated,water added, and the mixture extracted with ethyl acetate. The combinedorganic layers are washed with water, 10% potassium hydrogen sulfate,and brine, and then concentrated. The product is purified by flashchromatography.

[0510] Step 8B: Synthesis of tert-butyl methyl(S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-((tert-butyl)oxycarbonyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)-4-((phenylmehtoxycarbonylamino)butanoylamino)pentane-1,5-dioate

[0511] The product of 8a is dissolved in one volume of dichloromethaneand treated with excess triethylsilane and one volume of trifluoroaceticacid. The reaction is stirred under nitrogen for three hours and thenconcentrated to an oil. The triacid residue is dissolved indimethylformamide and treated with excess gamma-tert-butyl-alpha-methylglutamate, HBTU, HOBT, and diisopropylethylamine with stirring undernitrogen for 4-5 hours. The reaction is concentrated, partitioned intowater/ethyl acetate and extracted with more ethyl acetate. The combinedorganics are washed with water and brine and concentrated to an oil,which is purified by flash chromatography using dichloromethane/ethylacetate/methanol.

[0512] Step 8C: Synthesis of methyl(S,S,S,S,S,S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-2-(4-(N-(1,3-bis(N-(3-(N-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)-4-((phenylmethoxy)carbonylamino)butanoylamino)butanoate

[0513] The product of 8b is dissolved in one volume of dichloromethaneand treated with excess triethylsilane and one volume of trifluoroaceticacid. The reaction is stirred under nitrogen for three hours and thenconcentrated to an oil.

[0514] A threefold excess of the product of step 1F is treated in thesame fashion with trifluoroacetic acid and triethylsilane andconcentrated to an oil. The two residues are dissolved indimethylformamide, combined, and treated with HBTU, HOBT, anddiisopropylethylamine with stirring under nitrogen, followingdisappearance of starting material by HPLC. When complete, the reactionis concentrated, partitioned into water/ethyl acetate and extracted withmore ethyl acetate. The combined organics are washed with water andbrine and concentrated to an oil, which is purified by preparative HPLCusing a 0.1% trifluoroacetic acid/acetonitrile gradient to afford theproduct as a powder after lyophilization.

[0515] Step 8D: Synthesis of methyl(S,S,S,S,S,S,S,S)-2-(4-amino-4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)butanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)butanoate

[0516] The product of step 8C is dissolved in methanol with 10%palladium on carbon and 2 equivalents of acetic acid in a Parr bottle.The mixture is hydrogenated at 55 psi in a Parr shaker, following byHPLC until all the starting material has been reacted. The reaction isfiltered through Celite, concentrated, and the residual oil lyophilizedfrom water/acetonitrile to yield the product as a powder, to be useddirectly in the next step.

[0517] Step 8E: Conjugation of 8D with 7I

[0518] The product of step 8D is reacted with the product of step 7I asdescribed in the alternate synthesis of 7E to afford the product as asolid after preparative HPLC purification and lyophilization.

[0519] Step 8F: Synthesis of(S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-4-(2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)-4-carboxybutanoylamino)-4-carboxybutanoylamino)butanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)butanoic acid

[0520] The product of step 8E is dissolved in 2:1methanol/tetrahydrofuran and excess lithium hydroxide (3M solution)added. The solution is stirred, following by HPLC, until all the methylesters have been hydrolyzed. The reaction is quenched with solid citricacid, concentrated, and redissolved in one volume of dichloromethane.The solids are filtered and the filtrate treated with excesstriethylsilane and one volume of trifluoroacetic acid. The solution isstirred under nitrogen, following by HPLC, until all of the tert-butylesters have been hydrolyzed. The reaction mixture is concentrated anddirectly purified by preparative HPLC using 0.1% formicacid/acetonitrile gradient on a Zorbax C-8 column to afford the productafter lyophilization.

Example 9 Preparation of(S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

[0521]

[0522] Step 9A: Synthesis of N-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)(tert-butoxy)formamide

[0523] A solution of at least three equivalents of4,7,10-trioxa-1,13-tridecanediamine in tetrahydrofuran is cooled to 0°C., and a solution of one equivalent of di-tert-butyl dicarbonate inacetonitrile is added dropwise with stirring. The solution is stirredunder nitrogen overnight and then concentrated. The residue is dissolvedin ether and washed with five portions of saturated sodium chloride. Theorganic layer is dried over magnesium sulfate, filtered and concentratedto an oil, which is purified by flash chromatography to afford themonoamine.

[0524] Step 9B: Synthesis of tert-butyl3-(((3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)amino)methyl)-4-fluorobenzoate

[0525] The product of step 9A is treated with crudetert-butyl-4-fluoro-3(alpha-bromomethyl)benzoate, as described in step1A, to afford the product after flash chromatography.

[0526] Step 9C: Synthesis of methyl(S)-3-(N-(3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate

[0527] The product of step 9B is treated with Z-aspartic acid-β-methylester as described in step 1B, to afford the product after flashchromatography.

[0528] Step 9D: Synthesis of methyl(S)-3-amino-3-(N-(3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)propanoate

[0529] The product of step 9C is treated as in step 1C, and useddirectly in the following step.

[0530] Step 9E: Synthesis of methyl (S)-2-(2,5-diaza-9-((tert-butyl)oxycarbonyl-5-(3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0531] The product of step 9D is treated as in step 1D, to afford theproduct after flash chromatography.

[0532] Step 9F: Synthesis of(S)-2,5-diaza-5-(3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-3-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-9-carboxylicacid

[0533] The product of step 9E is treated as in step 1E, to afford theproduct after flash chromatography.

[0534] Step 9G: Synthesis of methyl(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0535] The product of step 9F is treated as in step 1F, to afford theproduct after flash chromatography.

[0536] Step 9H: Synthesis of(S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

[0537] The product of step 9G is treated as in step 2G, and the isolatedresidue then directly treated as in step 2H to afford the product afterpreparative HPLC and lyophilization.

Example 10 Preparation of(S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)propyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino) hexanoylamino)butanoic acid

[0538]

[0539] Step 10A: Synthesis of methyl(S)-2-(5-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0540] The product of step 9G is treated with trifluoroacetic acid andtriethylsilane in dichloromethane for 30 minutes and the reaction thenconcentrated to an oil. Toluene is added and the solution reconcentratedto an oil, which is used directly in the next step.

[0541] Step 10B: Synthesis of(S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)propyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)hexanoylamino)butanoic acid

[0542] The product of step 10A is treated in several steps as defined inexample 7, steps 7A-7F, substituting step 10A product for step 1Iproduct as a starting material in step 7A. The product is obtained as asolid after preparative HPLC purification and lyophilization.

Example 11 Synthesis of(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid

[0543]

[0544] Step 11A: Synthesis of tert-butyl methyl(S,S)-2-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)pentane-1,5-dioate

[0545] This process is carried out as in step 1G, except starting withalpha-methyl-gamma-tert-butylglutamate.

[0546] Step 11B: Synthesis of methyl(S,S)-4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)butanoate

[0547] The product of step 11A is dissolved in dichloromethane, followedby addition of trifluoroacetic acid (to form a 35% solution). This isstirred under nitrogen until the starting material and monoacid havedisappeared by HPLC, and then the solution is concentrated. The residueis dissolved in dimethylformamide along with 2.5 equivalents of1-amino-1-deoxysorbitol, 2.5 equivalents of HBTU, 2 equivalents ofhydroxybenzotriazole hydrate, and 3 equivalents diisopropylethylamine.The solution is stirred for two hours, concentrated, and the residuepurified by preparative HPLC.

[0548] Step 11C: Synthesis of(S,S)-4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)butanoic acid

[0549] The product of step 11B is dissolved in tetrahydrofuran/methanol(1:1) and treated with excess 3N aqueous lithium hydroxide. The reactionis followed by HPLC for disappearance of starting material. The reactionis concentrated, diluted with additional water, and purified by passagedown an acidic ion exchange column. The product fractions arelyophilized to afford the product as a solid.

[0550] Step 11D: Synthesis of methyl(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(phenylmethoxy)carbonylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0551] The product of step 2G is dissolved in dichloromethane andstirred with trifluoroacetic acid and triethylsilane for 15 minutes. Thesolution is concentrated, and the residue dissolved in dimethylformamidewith the product of step 11C, HBTU, hydroxybenzotriazole hydrate, anddiisopropylethylamine. The reaction is stirred, following by HPLC fordisappearance of starting materials. When complete, the solution isconcentrated and the residue purified by preparative HPLC. The productsolutions are lyophilized to afford the product.

[0552] Step 11E: Synthesis of methyl(S,S,S)-2-(5-(6-(2-(2-amino-4(-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)butanoylamino)-4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)butanoylamino)hexyl)-2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

[0553] The product of step 11D is treated as in step 1I, to afford theamine after concentration.

[0554] Step 11F: Synthesis of(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid

[0555] The product of step b 11E is reacted as in step 1J to afford theproduct after preparative HPLC purification.

[0556] Step 11G: Synthesis of(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

[0557] The product of step 11F is treated as in step 1K, to afford theproduct after preparative HPLC purification.

Example 12 Synthesis of(S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-carboxypropyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoic acid

[0558]

[0559] Step 12A: Synthesis of H-Asp(OtBu)-D-Phe-Lys(Cbz)-Arg(Mtr)-Gly-OH

[0560] This peptide is prepared using an Advanced Chemtech Model 90synthesizer using standard Fmoc protocols. The starting resin is4-[4-hydroxymethyl)-3-methoxy-phenoxy]butanoyl benzhydrylamine resinpreloaded with Fmoc-glycine (Fmoc-Gly-HMPB-BHA). Synthesis of theprotected linear peptide is achieved through sequential coupling (for 3hrs) of the amino acidsN-alpha-Fmoc-N⁹-4-methoxy-2,3,6-trimethylbenzenesulfonyl-1-arginine,N-alpha-Fmoc-N-epsilon-benzyloxycarbonyl-L-lysine, Fmoc-phenylalanine,and Fmoc-gamma-tert-butyl aspartic acid, using HBTU and HOBT as couplingagents. The couplings are carried out with five equivalents of aminoacid, HBTU, HOBT, and diisopropylethylamine in dimethylformamide. Fmocdeprotections are accomplished with 20% piperidine in DMF for 30minutes. The protected linear peptide is cleaved from the resin with 1%trifluoroacetic acid in dichloromethane and the peptide solutioncollected in 10% pyridine in methanol. The crude peptide is obtained byconcentrating the solvents in vacuo and triturating with diethyl ether.The peptide is purified by preparative HPLC and the product fractionsare lyophilized.

[0561] Step 12B: Synthesis ofcyclo{Lys(Cbz)-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}

[0562] HBTU (0.7 mmol) and hydroxybenzotriazole (0.5 mmol) are dissolvedin dimethylformamide (10 mL). The solution is warmed to 60° C. undernitrogen and a solution of the product of step 12 A (0.4 g) anddiisopropylethylamine (1.5 mmol) in dimethylformamide (10 mL) addedslowly. The solution is stirred at this temperature for 4 hours undernitrogen. The solution is concentrated and the residue triturated withethyl acetate. The resulting solids are washed with ethyl acetate anddried under vacuum to afford the product, which is used directly in thenext step.

[0563] Step 12C: Synthesis of cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}

[0564] The product of step 12 B is dissolved in 2-propanol and 10%palladium on carbon added with stirring. Hydrogen gas is gently bubbledinto the reaction mixture until all of the starting material is consumedby HPLC analysis. The reaction mixture is filtered through a bed ofCelite and the filtrate concentrated. The residue is not furtherpurified but used directly in the following step.

[0565] Step 12D: Synthesis of tert-butyl(S,S)-4-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-4-(((phenylmethoxy)carbonylamino)butanoate

[0566] The product of step 2F is dissolved in dichloromethane andtrifluoroacetic acid added (30% solution). The reaction is stirred 30minutes and concentrated. The residue is dissolved in dimethylformamideandN-carbobenzyloxy-gamma-tert-butyl-alpha-N-hydroxysuccinimidylglutamateadded, along with excess diisopropylethylamine. The reaction is stirredfor four hours and concentrated. The residue is purified by preparativeHPLC and the fractions lyophilized to afford the product as a solid.

[0567] Step 12E: Synthesis of(S,S)-4-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-4-(((phenylmethoxy)carbonylamino)butanoyl-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe} conjugate

[0568] The product of step 12D is dissolved in one volume ofdichloromethane, followed by one volume of trifluoroacetic acid and 5equivalents of triethylsilane. The solution is stirred for four hoursand concentrated. The residue is dissolved in dimethylformamidecontaining the product of step 12C, HBTU, and hydroxybenzotriazolehydrate. Diisopropylethylamine is added to this mixture with stirringunder nitrogen, following by HPLC for disappearance of the startingmaterials. When complete, the reaction is concentrated and the residuepurified by preparative HPLC. The product fractions are combined andlyophilized.

[0569] Step 12F: Synthesis of(S,S)-4-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-4-amino)butanoyl)-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}conjugate

[0570] The product of step 12E is treated as in step 8D. The product isnot further purified, but used directly in the next step.

[0571] Step 12G: Synthesis of tert-butyl(S,S,S,S)-4-(N-(1-N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}carbamoyl)propyl)carbamoyl-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclcododecyl)acetylamino)butanoate

[0572] The product of step 12F is treated as in step 8E to afford theproduct after preparative HPLC purification.

[0573] Step 12H: Synthesis of(S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-carboxypropyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclcododecyl)acetylamino)butanoic acid

[0574] The product of step 12G is dissolved in tetrahydrofuran andexcess lithium hydroxide added as a 3N solution in water. The solutionis stirred under nitrogen, following by HPLC for disappearance ofstarting material. When this is complete, the reaction is acidified with10% potassium hydrogen sulfate and concentrated. The residue isdissolved in neat trifluoroacetic acid containing thioanisole andstirred at room temperature under nitrogen, following the multipledeprotections by HPLC, until complete. The reaction is concentrated andthe crude residue purified by preparative HPLC.

[0575] The following procedure describe the synthesis ofradiopharmaceuticals of the present invention of the formula^(99m)Tc(VnA)(tricine)(phosphine), in which (VnA) represents avitronectin receptor antagonist compound of the present invention bondedto the Tc through a diazenido (—N═N—) or hydrazido (═N—NH—) moiety. Thediazenido or hydrazido moiety results from the reaction of thehydrazinonicotinamido group, present either as the free hydrazine orprotected as a hydrazone, with the Tc-99m. The other two ligands in theTc coordination sphere are tricine and a phosphine.

Examples 13-14 Synthesis of Complexes [^(99m)Tc (HYNIC-VnA) (tricine)(TPPTS)]

[0576] To a lyophilized vial containing 4.84 mg TPPTS, 6.3 mg tricine,40 mg mannitol, succinic acid buffer, pH 4.8, and 0.1% Pluronic F-64surfactant, was added 1.1 mL sterile water for injection, 0.2 mL (20 μg)of the appropriate HYNIC-conjugated vitronectin antagonist (VnA) indeionized water or 50% aqueous ethanol, and 0.2 mL of ^(99m)TcO₄ ⁻ (50±5mCi) in saline. The reconstituted kit was heated in a 100° C. water bathfor 15 minutes, and was allowed to cool 10 minutes at room temperature.A sample of the reaction mixture was analyzed by HPLC. The RCP resultsare listed in the table 1. TABLE 1 Analytical and Yield Data for^(99m)Tc (VnA) (tricine) (TPPTS) Complexes Ret. Time Example No. ReagentNo. (min) % Yield 13 2 8.9* 86 14 3 22.5** 46

Examples 15-22 Synthesis of ¹⁷⁷Lu and ⁹⁰Y Complexes

[0577] To a clean sealed 10 mL vial was added 0.5 mL of a solution ofthe appropriate conjugate (200 μg/mL in 0.25 M ammonium acetate buffer,pH 7.0), followed by 0.05-0.1 mL of gentisic acid (sodium salt, 10 mg/mLin 0.25 M ammonium acetate buffer, pH 7.0) solution, 0.3 mL of 0.25 Mammonium acetate buffer (pH 7.0), and 0.05 mL of ¹⁷⁷LuCl₃ solution (˜200mCi/mL) or ⁹⁰YCl₃ solution (100-200 mCi/mL) in 0.05 N HCl. The resultingmixture was heated at 100° C. for 35 min. After cooling to roomtemperature, a sample of the resulting solution was analyzed byradio-HPLC and ITLC. For ⁹⁰Y complexes, the sample has to be diluted15-20 fold before the radio-HPLC analysis. The ITLC method used GSsilica-gel paper strips and a 1:1 mixture of acetone and saline aseluant. The analytical and yield data are shown in Table 2. TABLE 2Analytical and Yield Data for Lu-177 and Y-90 Complexes Reagent Ex. Ret.Time Example No. No. Isotope (min) % RCP 15 1 ¹⁷⁷Lu 14.3 94 16 1 ⁹⁰Y14.0 92 17 1 ¹⁴⁹Pm 14.0 94 18 5 ¹⁷⁷Lu 14.1 94 19 5 ⁹⁰Y 14.7 93 20 5¹⁴⁹Pm 15.0 94 21 6 ¹⁷⁷Lu 17.1 94 22 6 ⁹⁰Y 17.4 84

[0578] HPLC Method

[0579] Column: Zorbax C18 , 25 cm×4.6 mm

[0580] Flow rate: 1.0 mL/min

[0581] Solvent A: 25 mM sodium phosphate buffer, pH 6.0

[0582] Solvent B: 100% CH₃CN

[0583] Gradient I t (min) 0 20 21 30 31 40 % Solvent B 0 20 60 60 0 0

[0584] The identity of the Lu-177 complexes of Examples 15, 18, and 21were further confirmed by LC-MS. The MS data are shown in Table 3. TABLE3 Mass Spec. Data for Lu-177 Complexes Example No. Formula Atomic WeightM + H⁺ 15 C₅₀H₆₅LuN₁₂O₁₇ 1280.4 1282.0 18 C₅₂H₆₇LuN₁₂O₁₇ 1306.4 1307.321 C₄₆H₅₈LuN₁₁O₁₇ 1163.4 1164.2

Example 23 Synthesis of the ¹¹¹In Complex of the Conjugate of Example 1

[0585] To a lead shielded and closed autosampler vial was added 65 μg ofthe conjugate of Example 1 and 1.5 mg gentisic acid, sodium saltdissolved in 65 μL ammonium acetate buffer (0.4 M, pH 4.7) followed bythe addition of 1.8 mCi , 15 μL In-111 in 0.05 N HCl (specific activity:36 μg/mCi) . The reaction mixture was heated at 70-80° C. for 60 min andanalyzed by HPLC and ITLC. The radiolabeling yield was 91% and theretention time was 9.8 min.

[0586] HPLC Method

[0587] Column: Zorbax C18 , 25 cm×4.6 mm

[0588] Flow rate : 1.0 mL/min

[0589] Solvent A: 10 mM sodium phosphate buffer, pH 6.0

[0590] Solvent B : 100% CH₃CN

[0591] Gradient I t (min) 0 20 21 30 31 40 % Solvent B 5 20 60 60 5 5

[0592] The ITLC method used GS silica-gel paper strips and a 1:1 mixtureof acetone and saline as eluant.

Examples 24-25 Synthesis of the ¹¹¹In Complex of the Conjugates ofExample 5 and 6

[0593] To a lead shielded and closed autosampler vial was added 100 μgof the appropriate conjugate of the present invention dissolved in 100μL ammonium acetate buffer (0.2 M, pH 4.7) followed by 2.3 mCi, 25 μLIn-111 in 0.05 N HCl. The solutions were heated at 100° C. for 30 minand analyzed by HPLC and ITLC. The radiolabeling yield for Example 24was 76% and the retention time was 9.4 min. The radiolabeling yield forExample 25 was 87% and the retention time was 17.2 min.

[0594] The ITLC method used GS silica-gel paper strips and a 1:1 mixtureof acetone and saline as eluant.

[0595] HPLC Method (Example 24)

[0596] Column: Zorbax C18 , 25 cm×4.6 mm

[0597] Flow rate: 1.0 mL/min

[0598] Solvent A: 10 mM sodium phosphate buffer, pH 6.0

[0599] Solvent B: 100% CH₃CN

[0600] Gradient I t (min) 0 20 21 30 31 40 % Solvent B 5 20 60 60 5 5

[0601] HPLC Method (Example 25)

[0602] Column: Zorbax C18 , 25 cm×4.6 mm

[0603] Flow rate: 1.0 mL/min

[0604] Solvent A: 0.1% TFA in water

[0605] Solvent B : 100% CH₃CN

[0606] Gradient I t (min) 0 20 21 30 31 40 % Solvent B 5 20 60 60 5 5

Example 26 Preparation of sodium1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid-dodecoanoate conjugate

[0607]

[0608] Step 26A: Synthesis of sodium1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid-dodecoanoate conjugate

[0609] 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine monosodium salt(DPPE) (1.25 g, 0.5 mmol) is dissolved under nitrogen in chloroform (15mL) along with disuccinimidyl dodecanoate (0.212 g, 0.5 mmol and theproduct of step 4A (367 mg, 0.5 mmol). They are stirred for 5 minutes,when sodium carbonate (0.5 mmol) and sodium sulfate (0.5 mmol) is added.The reaction is stirred 18 hrs, filtered, and concentrated. The residueis purified to obtain the title compound.

[0610] Step 26B: Preparation of contrast agent composition

[0611] The product of step 13A is admixed with three other lipids,1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline, andN-(methoxypolyethylene glycol5000)carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine inrelative amounts of 1 wt %:6 wt %:54 wt %:41 wt %. An aqueous solutionof this lipid admixture (1 mg/mL), sodium chloride (7 mg/mL), glycerin(0.1 mg/mL), and propylene glycol (0.1 mL/mL) at pH 6-7 is then preparedin a 2 cc glass vial. The air in the vial is evacuated and replaced withperfluoropropane and the vial is sealed. The ultrasound contrast agentcomposition is completed by agitating the sealed vial in a dentalamalgamator for 30-45 seconds to form a milky white solution.

Example 27 Preparation ofDPPE-PEG₃₄₀₀-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid]-dodecoanoate conjugate

[0612]

[0613] Step 27A: Synthesis ofω-amino-PEG₃₄₀₀-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid]

[0614] A solution of N-Boc-ω-amino-PEG₃₄₀₀-succinimidyl ester (1 mmol)and the product of step 4A (1 mmol) in DMF (15 mL) is treated withdiisopropylethylamine (3 mmol) and stirred under nitrogen for 18 hr. Thesolution is concentrated and the residue dissolved in dichloromethane (8mL) to which trifluoroacetic acid (6 mL) is added. The solution isstirred for 30 minutes, and then concentrated under vacuum. The productis isolated by trituration with diethyl ether.

[0615] Step 27B: Synthesis ofDPPE-PEG₃₄₀₀-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid]-dodecoanoate conjugate

[0616] A solution of disuccinimidyl dodecanoate (0.5 mmol), DPPE (0.5mmol), and the product of step 14A (0.5 mmol) are added to 10 mLchloroform with stirring under nitrogen. Sodium carbonate (1 mmol) andsodium sulfate (1 mmol) are added and the solution is stirred at roomtemperature for 18 hrs. The reaction is filtered, the solventconcentrated, and the residue purified to obtain the title compound.

[0617] Step 27C: Preparation of contrast agent composition

[0618] The product of step 14B is admixed with three other lipids,1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline, andN-(methoxypolyethylene glycol5000)carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine inrelative amounts of 1 wt %:6 wt %:54 wt %:41 wt %. An aqueous solutionof this lipid admixture (1 mg/mL), sodium chloride (7 mg/mL), glycerin(0.1 mg/mL), and propylene glycol (0.1 mL/mL) at pH 6-7 is then preparedin a 2 cc glass vial. The air in the vial is evacuated and replaced withperfluoropropane and the vial is sealed. The ultrasound contrast agentcomposition is completed by agitating the sealed vial in a dentalamalgamator for 30-45 seconds to form a milky white solution.

Example 28 Preparation of[(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)carbamoyl)propyl)carbamoyl]-□-amino-PEG₃₄₀₀-dodecanoate-DPPEconjugate

[0619]

[0620] Step 28A: Synthesis of[(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)carbamoyl)propyl)carbamoyl]-ω-amino-PEG₃₄₀₀

[0621] The product of step 4B (1 mmol) was deprotected as described instep 4C and added to a solution of N-Boc-ω-amino-PEG₃₄₀₀-succinimidylester (1 mmol) in DMF (15 mL). Diisopropylethylamine (3 mmol) is addedand the solution stirred under nitrogen for 18 hr. The solution isconcentrated and the residue dissolved in dichloromethane (8 mL) towhich trifluoroacetic acid (6 mL) is added. The solution is stirred for30 minutes, and then concentrated under vacuum. The product is isolatedby trituration with diethyl ether.

[0622] Step 28B: Synthesis ofDPPE-PEG₃₄₀₀-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid]-dodecoanoate conjugate

[0623] A solution of disuccinimidyl dodecanoate (0.5 mmol), DPPE (0.5mmol), and the product of step 15A (0.5 mmol) are added to 10 mLchloroform with stirring under nitrogen. Sodium carbonate (1 mmol) andsodium sulfate (1 mmol) are added and the solution is stirred at roomtemperature for 18 hrs. The reaction is filtered, the solventconcentrated, and the residue purified to obtain the title compound.

[0624] Step 28C: Preparation of contrast agent composition

[0625] The product of step 15B is admixed with three other lipids,1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline, andN-(methoxypolyethylene glycol5000)carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine inrelative amounts of 1 wt %:6 wt %:54 wt %:41 wt %. An aqueous solutionof this lipid admixture (1 mg/mL), sodium chloride (7 mg/mL), glycerin(0.1 mg/mL), and propylene glycol (0.1 mL/mL) at pH 6-7 is then preparedin a 2 cc glass vial. The air in the vial is evacuated and replaced withperfluoropropane and the vial is sealed. The ultrasound contrast agentcomposition is completed by agitating the sealed vial in a dentalamalgamator for 30-45 seconds to form a milky white solution.

Example 29 Synthesis of4-[N-(3-{(2R)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl](4S)-4-[(4S)-4-(N-{(1S)-1-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-3-carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}butanoylamino]butanoicacid

[0626]

[0627] Step 29A: Synthesis of

[0628] The product of step 1F (100 mg, 0.172 mmol) was dissolved indichloromethane (4 mL) and treated with trifluoroacetic acid (4 mL) andtriethylsilane (160 uL) under nitrogen. The reaction was stirred for 25minutes and concentrated under vacuum, treated with toluene (5 mL) andreconcentrated. The residue was dissolved in DMF (2 mL) and treated withtert-butyl 2,5-dioxopyrrolidinyl(2S)-2-[(phenylmethoxy)carbonyl-amino]pentane-1,5-dioate (85 mg, 0.19mmol) and di;sopropylethylamine (135 uL, 0.775 mmol). The mixture wasstirred under nitrogen for 1 hour and then partitioned into ethylacetate/water (1:1, 100 mL). The layers were separated and the aqueouslayer extracted with two more portions of ethyl acetate. The combinedorganic layer was washed with water and brine, dried over sodiumsulfate, filtered, and concentrated to afford the product as a pale oilwhich solidified under vacuum (145 mg, 105%). This was used directly inthe next step. LRMS (ES): 798.4 [M+H]⁺, 100%

[0629] Step 29B: Synthesis of

[0630] The product of step 29A is deprotected as in step 6B to afford animpure oil. This was purified by preparative HPLC (Vydac C18, 2.25×25cm, 90% acetonitrile/water/0.1% TFA; 5-55% B over 25 minutes), theproduct fractions combined, frozen, and lyophilized to afford theproduct as the bis-TFA salt (100 mg, 97%). LRMS (ES): 664.4 ([M+H]+,20%), 333.0 ([M+2H]+2, 100%).

[0631] Step 29C: Synthesis of bis-2,3,5,6-tetrafluorophenyl(2S)-2-[(tert-butoxy)carbonylamino]pentane-1,5-dioate

[0632] Boc-Glutamic acid (4.0 g, 16.2 mmol) was dissolved in DMF (60 mL)with 2,3,5,6-tetrafluorophenol (6.5 g, 39 mmol). To this was added(3-dimethylaminopropyl)ethyl carbodiimide hydrochloride (7.4 g, 39 mmol)and the solution was stirred 18 hr. The reaction was concentrated andthe residue partitioned between ethyl acetate and water. The aqueouslayer was extracted three times with ethyl acetate, and the combinedorganic layer was washed with 0.1N HCl, water, and brine. It wasconcentrated to a white solid which was washed with two portions ofacetonitrile and dried under vacuum to afford the product as a whitesolid (6.2 g, 70%) with mp=123.5-124.5C. LRMS: 566.0 [M+Na]⁺. ¹HNMR(600.1343 MHz, CDCl₃): 7.02 (m, 2H), 5.14 (m, 1H), 4.80 (m, 1H), 2.92(m, 2H), 2.53 (m, 1H), 2.80 (m, 1H), 1.47 (s, 9H).

[0633] Step 29D: Synthesis of

[0634] The product of step 29B (95 mg, 94 umol) was treated with theproduct of 29C (24.4 mg, 45 umol) and diisopropylethylamine (99 uL, 570umol) in DMF and allowed to stir under nitrogen for 20 hr. The reactionwas concentrated, water added and extracted three times with ethylacetate. The combined organics were washed with 0.1N NaOH, water, andbrine, dried over magnesium sulfate, filtered and concentrated to awhite film (63 mg, 91%) which was not further purified but used directlyin the next step. LRMS (ES): 1538.1 ([M+H]⁺, 5%), 770.0 ([M+2H]⁺²,100%), 514.0 ([M+3H]⁺³, 25%).

[0635] Step 29E: Synthesis of

[0636] The product of step 29D (60 mg, 39 umol) was dissolved indichloromethane (2.5 mL) under nitrogen. Trifluoroacetic acid (2.5 mL)and triethylsilane were added (100 uL) and the solution stirred for 1.5hr. The reaction was concentrated and chased with toluene (2×5 mL). Theresidue was dissolved in THF/methanol (1:1, 3 mL) and treated with a 3Nsolution of lithium hydroxide in water (260 uL, 390 umol). Afterstirring for 12 hours, another aliquot of lithium hydroxide (130 uL) wasadded and stirring continued for five hours. The reaction was acidifiedwith 0.1N HCl to pH=2 and concentrated. Purification by preparative HPLC(Vydac C18, 2.25×25 cm, 90% acetonitrile/water/0.1% TFA; 5-35% B over 50minutes), combining product fractions, and lyophilizing afforded theproduct as a white solid (23 mg 45%). LRMS (ES): 1298.4 ([M+H]⁺, 10%),649.9 ([M+2H]⁺², 30%), 433.6 ([M+3H]⁺³, 100%).

[0637] Step 29F: Synthesis of

[0638] The product of step 29E (20 mg, 14.1 umol) was dissolved in dryDMF (0.5 mL) with diisopropylethylamine (15 μL, 85 umol) under nitrogen.In another flask under nitrogen, DOTA(OtBu)₃-OH (17 mg, 21 umol) wasdissolved in DMF with diisopropylethylamine (15 uL, 85 umol) and HBTU(6.7 mg, 18 umol) and stirred 10 minutes. The activated DOTA solution isadded in one portion to the amine and stirred for 30 minutes. Thereaction was concentrated and purified by preparative HPLC (Vydac C18,2.25×25 cm, 90% acetonitrile/water/0.1% TFA; 15-535% B over 50 minutes),combining product fractions, and lyophilizing afforded the product as awhite solid (8 mg, 30%). LRMS (ES): 1853.0 [M+H]⁺,

[0639] Step 29G: Synthesis of

[0640] The product of step 29F (7 mg) was dissolved in trifluoroaceticacid (2 mL) with triethylsilane (200 uL) under nitrogen and stirred for30 minutes. The solution was concentrated and purified by prep HPLC(Vydac C18, 2.25×25 cm, 50% acetonitrile/water/0.1% formic acid; 15-35%B over 50 minutes). The product fractions were combined and lyophilizedto afford a white solid (2 mg). LRMS (ES) : 1684.6 ( [M+H]⁺, 5%), 843.0( [M+2H]⁺², 50%), 562.5 ([M+3H]⁺³, 100%).

Example 30 Synthesis of2-(4-{3-[(6-{[(1E)-1-aza-2-(2-sulfophenyl)vinyl]amino}(3-pyridyl))carbonylamino]propyl} (2S)-7-(N-[2-(amidinoamino)ethyl]-N-methylcarbamoyl}-3-oxo-1H,2H,5H-benzo[f]1,4-diazepin-2-yl)aceticacid

[0641]

[0642] Step 30A: Synthesis of

[0643] The product from step 1E (100 mg, 220 umol),N-[2-(methylamino)ethyl](phenylmethoxy)carboxamide hydrochloride (57 mg,230 umol), (3-dimethylaminopropyl)ethyl carbodiimide hydrochloride (51mg, 264 umol), and HOBT (31.2 mg, 230 umol) were dissolved in DMF (2.2mL) under nitrogen and the solution was stirred 18 hr. The reaction wasconcentrated and the residue partitioned between ethyl acetate andwater. The aqueous layer was extracted three times with ethyl acetate,and the combined organic layer was washed with 0.1N HCl, water, andbrine. It was dried over sodium sulfate, filtered, and concentrated to aclear oil, which was purified by flash chromatography (2% methanol/ethylacetate). Product fractions were combined and concentrated to yield theproduct as an oil (110 mg, 80%). LRMS (ES): 626.4 ([M+H]⁺, 100%), 648.4([M+Na]⁺, 100%) 1273.7 ([2M+Na]⁺, 15%).

[0644] Step 30B: Synthesis of

[0645] The product of Step 30A (110 mg) was treated as in step 1I toafford the product (98 mg, 100%) as a white solid. LRMS (ES): 492.4([M+H]⁺, 100%), 514.4 ([M+Na]⁺, 30%)

[0646] Step 30C: Synthesis of

[0647] The product of step 30B (45 mg, 92 umol) was dissolved in DMF(0.6 mL) with diisopropylethylamine (33 uL, 185 umol), andtert-butyl-2-aza-3-[(tert-butoxy)carbonylamino]-3-methylthioprop-2-enoate(26.6 mg, 92 umol). Mercuric chloride (25 mg, 92 umol) was added and thereaction stirred 75 min. It was then diluted with ethyl acetate,filtered through Celite, and the solids rinsed. The combined filtratewas washed with water and brine, dried over sodium sulfate, filtered andconcentrated to afford a crude oil, which was purified by prep HPLC(Vydac C18, 2.25×25 cm, 90% acetonitrile/water/0.1% TFA; 10-70% B over30 minutes). The product fractions were combined and lyophilized toafford the product as a white solid (18 mg, 30%) which as a mixture ofproduct and deprotected material, which was used directly in the nextreaction. LRMS (ES): 734.4 [M+H]⁺, 634.4 [M-Boc+H]⁺.

[0648] Step 30D: Synthesis of

[0649] The product of step 30C (16 mg, 22 umol) was treated as in step16E, and purified by prep HPLC (Vydac C18, 2.25×25 cm, 90%acetonitrile/water/0.1% TFA; 0-25% B over 30 minutes). The productfractions were combined and lyophilized to afford the product as a whitesolid (6 mg, 52%). LRMS (ES): 420.2 ([M+H]⁺, 30%) 210.7 ([M+2H]⁺²,100%).

[0650] Step 30E: Synthesis of

[0651] The product of step 30D is treated as in step 3E, purified byprep HPLC and lyophilized to afford the product.

Example 31 Synthesis of2-[9-(N-{6-[(6-{[(1E)-1-aza-2-(2-sulfophenyl)vinyl]amino}(3-pyridyl))carbonylamino]hexyl}-N-(benzimidazol-2-ylmethyl)carbamoyl)(5S)-5,6,11-trihydro-dibenzo[b,e][7]annulen-5-yl]aceticacid

[0652]

[0653] Step 31A: Synthesis of

[0654] The product of step 3A (300 mg, 0.66 mmol) and6-[(ethoxycarbonyl)methyl]-5,6,11-trihydrodibenzo[a,d][7]annulene-2-carboxylic acid (215 mg, 0.66 mmol, preparedaccording to W. H. Miller et al., Bioorg. Med. Chem. Lett., 9(1999)1807-1812) are treated as in step 3B to yield the product after flashchromatography.

[0655] Step 31B: Synthesis of

[0656] The product of step 31A (100 mg, 0.15 mmol) is dissolved THF (3mL) with lithium hydroxide (3N solution in water, 0.5 mL, 1.5 mmol) andstirred, monitoring for disappearance of starting material by HPLC. Whenthe reaction is complete, the solution is acidified to pH=2 with 0.1NHCl and the resulting solids are filtered and dried under vacuum toafford the product, which is used directly in the following step.

[0657] Step 31C: Synthesis of

[0658] The product of step 31B is treated as in step 3C to afford theproduct as a solid after lyophilization.

[0659] Step 31D: Synthesis of

[0660] The product of step 31C is treated as in step 3E to afford theproduct as a yellow solid after prep HPLC purification andlyophilization.

Example 32 Synthesis of(2S)-2-[(2S)-4-(N-{(1S)-3-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-1-carboxypropyl}carbamoyl)-2-[(2S)-2-((2S)-4-carboxy-2-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}butanoylamino)-4-carboxybutanoylamino]butanoylamino]-4-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl] butanoic acid

[0661]

[0662] Step 32A: Synthesis of tert-butyl methyl(2S)-2-[(2S)-4-(N-{(1S)-3-[(tert-butyl)oxycarbonyl]-1-(methoxycarbonyl)propyl}carbamoyl)-2-[(phenylmethoxy)carbonylamino]butanoylamino]pentane-1,5-dioate

[0663] Cbz-glutamic acid (1 g, 3.56 mmol) was dissolved in DMF (20 mL)along with H-Glu(OtBu)OMe-HCl (1.9 g, 7.5 mmol), HBTU (3.4 g, 8.9 mmol),HOBT (1.01 g, 7.5 mmol), and diisopropylethylamine (2.2 mL, 12.5 mmol)under nitrogen. The reaction was stirred for 18 hours, concentrated, andpartitioned between water and ethyl acetate. The solids were filteredand the filtrate layers separated. The aqueous layer was extracted withethyl acetate and the combined organic layers washed with 10% sodiumcarbonate, water, 10% potassium hydrogen sulfate, water, and brine. Thesolution was dried over sodium sulfate, filtered, and concentrated toafford a golden oil which was purified by flash chromatography (4:1dichloromethane/ethyl acetate). The product fractions were combined andconcentrated to afford the product as a clear oil (1.3 g, 54%) whichsolidified under vacuum. LRMS (ES): 680.5 ([M+H]⁺, 100%), 702.5([M+Na]⁺, 20%)

[0664] Step 32B: Synthesis of methyl(2S)-2-[(2S)-4-(N-{(1S)-3-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-[(methoxycarbonyl)methyl]-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-1-(methoxycarbonyl)propyl}carbamoyl)-2-[(phenylmethoxy)carbonylamino]butanoylamino]-4-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-[(methoxycarbonyl)methyl]-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]butanoate

[0665] The product of step 1F (104 mg, 180 umol) was dissolved indichloromethane (2 mL) and trifluoroacetic acid (1 mL) added withstirring under nitrogen. The solution was stirred for 1 hour,concentrated under vacuum, and reconcentrated twice with toluene toafford the benzodiazepine amine as an oil which was used directly below.

[0666] The product of step 32A (43 mg, 63 umol) was dissolved indichloromethane (0.5 mL) and trifluoroacetic acid (0.9 mL) added withstirring under nitrogen. The solution was stirred for 2 hours,concentrated under vacuum, and reconcentrated twice with toluene toafford the dicarboxylic acid as an oil which was used directly below.

[0667] Both of these products were dissolved in DMF (1.5 mL) undernitrogen, and HBTU (60 mg, 150 umol), HOBT (20 mg, 140 umol), anddiisopropylethylamine (180 uL, 1.1 mmol) added. The solution was stirredfor 18 hours, concentrated, and the residue purified by preparative HPLC(Vydac C18, 2.12×25 cm, 90% acetonitrile/water/0.1% TFA; 10-55% B over25 minutes). The product fractions were combined and lyophilized toafford the product as a white solid (84 mg, 69%). LRMS (ES): 1488.7([M+H]⁺, 10%), 745.1 ([M+2H]⁺², 100%), 497.3 ([M+3H]⁺³, 100%)

[0668] Step 32C: Synthesis of tert-butyl(4S)-4-[N-((1S)-1-{N-[(1S)-1,3-bis(N-{(1S)-3-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-[(methoxycarbonyl)methyl]-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-1-(methoxycarbonyl)propyl}carbamoyl)propyl]carbamoyl}-3-[(tert-butyl)oxycarbonyl]propyl)carbamoyl]-4-[(phenylmethoxy)carbonylamino]butanoate

[0669] The product of step 32B (70 mg, 47 umol) was dissolved inmethanol (5 mL) and added to 10% palladium on carbon (40 mg) suspendedin methanol (5 mL) under nitrogen in a pressure bottle. The slurry washydrogenated at 55 psi on a Parr apparatus for two hours, additionalcatalyst (35 mg) added, and repressurized. The hydrogenation wascontinued for an additional 3 hours, at which time the reaction wasfiltered through Celite, rinsed with methanol, and the combined filtrateconcentrated to a clear oil (49 mg). This was dissolved in dry DMF (1.5mL), along with the product of step 1G (22 mg, 42 umol), HBTU (18 mg, 46umol), HOBT (6.5 mg, 42 umol), and diisopropylethylamine (9 uL, 52 umol)in a flame-dried flask under nitrogen. The reaction was stirred for 5.5hours, concentrated, and the residue purified by preparative HPLC (VydacC18, 2.12×25 cm, 90% acetonitrile/water/0.1% TFA; 10-70% B over 30minutes). The product fractions were combined and lyophilized to affordthe product as a white solid (32 mg, 48%). LRMS (ES): 1859.2 ([M+H]⁺,5%), 930.1 ([M+2H]⁺², 85%), 620.8 ([M+3H]⁺³, 100%)

[0670] Step 32D: Synthesis of tert-butyl(4S)-4-[N-((1S)-1-{N-[(1S)-1,3-bis(N-{(1S)-3-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-[(methoxycarbonyl)methyl]-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-1-(methoxycarbonyl)propyl}carbamoyl)propyl]carbamoyl}-3-[(tert-butyl)oxycarbonyl]propyl)carbamoyl]-4-[2-(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)oxycarbonyl]methyl}cyclododecyl)acetylamino]butanoate

[0671] The product of step 32C (30 mg, 13.6 umol) was dissolved inmethanol (6 mL) and added to 10% palladium on carbon (45 mg) in methanol(6 mL) and acetic acid (120 uL). The mixture was hydrogenated for 6.5hours at 55 psi, filtered, concentrated, and the residue dissolved in50% water/acetonitrile (20 mL), frozen, and lyophilized to yield a whitepowder (20.6 mg). This was dissolved in dry DMF (1 mL) along with HBTU(20 mg, 53 umol), HOBT (2.3 mg, 15 umol), and diisopropylethylamine (15uL, 75 umol). The reaction was stirred for 1.5 hours, concentrated, andthe residue purified by preparative HPLC (Vydac C18, 2.12×25 cm, 90%acetonitrile/water/0.1% TFA; 50-75% B over 26 minutes). The productfractions were combined and lyophilized to afford the product as a whitesolid (9.6, 30% LRMS (ES): 2279.5 ([M+H]⁺, 10%), 1140.3 ([M+2H]⁺², 20%),760.8 ([M+3H]⁺³, 100%). HRMS: Calculated for C₁₁₃H₁₆₄N₂₁O_(29—)2279.004;Found—2279.198.

[0672] Step 32E: Synthesis of(2S)-2-[(2S)-4-(N-{(1S)-3-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo{1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-1-carboxypropyl}carbamoyl}-2-[(2S)-2-((2S)-4-carboxy-2-(2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino)butanoylamino)-4-carboxybutanoylamino]butanoylamino]-4-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo (1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl) carbamoyl]butanoic acid

[0673] The product of step 32D (8 mg, 3 umol) was dissolved inmethanol/THF (1:1, 600 uL) and lithium hydroxide (3N solution, 10 uL, 30umol) added. Additional aliquots of lithium hydroxide were added (20 uLat 1 hr, 3 hr, and 5 hr) and the reaction worked up at 6 hours. It wasacidified with trifluoroacetic acid, concentrated, and the residuedissolved in dichloromethane (0.6 mL) along with trifluoroacetic acid(0.8 mL) and triethylsilane (100 uL). The solution was stirred undernitrogen for 20 hours, concentrated, and the residue purified bypreparative HPLC (Vydac C18, 2.12×25 cm, 90% acetonitrile/water/0.1%TFA; 12-23% B over 50 minutes). The product fraction was lyophilized toafford the product as a white solid (2.1 mg, 38%). LRMS (ES): 1942.6([M+H]⁺, 5%), 971.9 ([M+2H]⁺², 15%), 648.4 ([M+3H]⁺³, 55%), 486.6([M+4H]⁺⁴, 100%).

Example 33 Synthesis of3-(7-[3-(amidinoamino)propyl]-2,5-dioxo-1-{[4-(3-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}propoxy)phenyl]methyl}-3H-benzo[f]1,4-diazaperhydroepin-4-yl)propanoic acid

[0674]

Example 34 Synthesis of3-(8-[3-(amidinoamino)propyl]-2,5-dioxo-1-{[4-(3-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}propoxy)phenyl]methyl}-3H-benzo[f]1,4-diazaperhydroepin-4-yl)propanoicacid

[0675]

[0676] Compounds in Example 33 and Example 34 are prepared by thesynthetic route shown in Scheme I.

[0677] The procedure described below elucidates Scheme I.

[0678] Step I: 7—Substituted or 8-substituted IB may be prepared via thealkylation of ethyl3-(7-{3-[(tert-butoxy)carbonylamino]propyl}-2,5-dioxo-1H,3H-benzo[f]1,4-diazaperhydroepin-4-yl)propanoateor ethyl3-(8-{3-[(tert-butoxy)carbonylamino]propyl}-2,5-dioxo-1H,3H-benzo[f]1,4-diazaperhydroepin-4-yl)propanoate(IA) with 4-(2,4-dimethoxybenzyloxy)benzylbromide in the presence ofbase, followed by removal of the 2,4-dimethoxybenzyl protecting group.Alternately, 7-substituted or 8-substituted IB may be prepared from1-aryl-6-iodoisatoic anhydride and 1-aryl-7-iodoisatoic anhydride bymethods known in the art (McDowell, R. S. et al, J. Amer. Chem. Soc.,1994, 116, 5077-5083 and Blackburn, B. et al, PCT Intl. Appl., WO9308174 A1 19930429 (CAS: 120:217745)). Step II: Compound IC is preparedby the alkylation of the hydroxyl group in IB with3-Cbz-aminopropylbromide in the presence of base such as NaH in asolvent such as DMF. Step III: Intermediate ID is prepared by thedeprotection of the Boc group with either trifluoroacetic acid orHCl-ethyl acetate followed by treatment of the intermediate withformamidinosulfonic acid in the presence of base (eg. 5% KHCO3).

[0679] Step IV: Removal of the benzyloxycarbonyl group (Z, Cbz) isachieved by hydrogenolysis (Pd/C) or TFA/triethylsilane to give IE.

[0680] Step V: The title compound IF is prepared by the conjugation ofIE with DO3A-tri-t-butyl ester (Macrocyclics), followed by base and TFAhydrolyses of the ethyl and t-butyl esters, respectively. The desiredcompound is purified by reversed phase preparative HPLC.

Utility

[0681] The pharmaceuticals of the present invention are useful forimaging angiogenic tumor vasculature, therapeutic cardiovascularangiogenesis, and cardiac pathologies associated with the expression ofvitronectin receptors in a patient or for treating cancer in a patient.The radiopharmaceuticals of the present invention comprised of a gammaray or positron emitting isotope are useful for imaging of pathologicalprocesses involving angiogenic neovasculature, including cancer,diabetic retinopathy, macular degeneration, restenosis of blood vesselsafter angioplasty, and wound healing, as well as atherosclerotic plaque,myocardial reperfusion injury, and myocardial ischemia, stunning orinfarction. The radiopharmaceuticals of the present invention comprisedof a beta, alpha or Auger electron emitting isotope are useful fortreatment of pathological processes involving angiogenic neovasculature,by delivering a cytotoxic dose of radiation to the locus of theangiogenic neovasculature. The treatment of cancer is affected by thesystemic administration of the radiopharmaceuticals resulting in acytotoxic radiation dose to tumors.

[0682] The compounds of the present invention comprised of one or moreparamagnetic metal ions selected from gadolinium, dysprosium, iron, andmanganese, are useful as contrast agents for magnetic resonance imaging(MRI) of pathological processes involving angiogenic neovasculature, aswell as atherosclerotic plaque, myocardial reperfusion injury, andmyocardial ischemia, stunning or infarction.

[0683] The compounds of the present invention comprised of one or moreheavy atoms with atomic number of 20 or greater are useful as X-raycontrast agents for X-ray imaging of pathological processes involvingangiogenic neovasculature, as well as atherosclerotic plaque, myocardialreperfusion injury, and myocardial ischemia, stunning or infarction.

[0684] The compounds of the present invention comprised of an echogenicgas containing surfactant microsphere are useful as ultrasound contrastagents for sonography of pathological processes involving angiogenicneovasculature, as well as atherosclerotic plaque, myocardialreperfusion injury, and myocardial ischemia, stunning or infarction.

[0685] Representative compounds of the present invention were tested inthe following in vitro assays and in vivo models and were found to beactive.

[0686] Immobilized Human Placental α_(v)β₃ Receptor Assay

[0687] The assay conditions were developed and validated using[I-125]vitronectin. Assay validation included Scatchard format analysis(n=3) where receptor number (Bmax) and Kd (affinity) were determined.Assay format, is such that compounds are preliminarily screened at 10and 100 nM final concentrations prior to IC50 determination. Threestandards (vitronectin, anti-α_(v)β₃ antibody, LM609, and anti-α_(v)β₅,P1F6) and five reference peptides have been evaluated for IC50determination. Briefly, the method involves immobilizing previouslyisolated receptors in 96 well plates and incubating overnight. Thereceptors were isolated from normal, fresh, non-infectious (HIV,hepatitis B and C, syphilis, and HTLV free) human placenta. The tissuewas lysed and tissue debris removed via centrifugation. The lysate wasfiltered. The receptors were isolated by affinity chromatography usingthe immobilized α_(v)β₃ antibody. The plates are then washed 3×with washbuffer. Blocking buffer is added and plates incubated for 120 minutes atroom temperature. During this time compounds to be tested and[I-125]vitronectin are premixed in a reservoir plate. Blocking buffer isremoved and compound mixture pipetted. Competition is carried out for 60minutes at room temperature. Unbound material is then removed and wellsare separated and counted via gamma scintillation.

[0688] Oncomouse® Imaging

[0689] The study involves the use of the c-Neu Oncomouse® and FVB micesimultaneously as controls. The mice are anesthetized with sodiumpentobarbital and injected with approximately 0.5 mCi ofradiopharmaceutical. Prior to injection, the tumor locations on eachOncomouse® are recorded and tumor size measured using calipers. Theanimals are positioned on the camera head so as to image the anterior orposterior of the animals. 5 Minute dynamic images are acquired seriallyover 2 hours using a 256×256 matrix and a zoom of 2×. Upon completion ofthe study, the images are evaluated by circumscribing the tumor as thetarget region of interest (ROI) and a background site in the neck areabelow the carotid salivary glands.

[0690] This model can also be used to assess the effectiveness of theradiopharmaceuticals of the present invention comprised of a beta, alphaor Auger electron emitting isotope. The radiopharmaceuticals areadministered in appropriate amounts and the uptake in the tumors can bequantified either non-invasively by imaging for those isotopes with acoincident imageable gamma emission, or by excision of the tumors andcounting the amount of radioactivity present by standard techniques. Thetherapeutic effect of the radiopharmaceuticals can be assessed bymonitoring the rate of growth of the tumors in control mice versus thosein the mice administered the radiopharmaceuticals of the presentinvention.

[0691] This model can also be used to assess the compounds of thepresent invention comprised of paramagnetic metals as MRI contrastagents. After administration of the appropriate amount of theparamagnetic compounds, the whole animal can be placed in a commerciallyavailable magnetic resonance imager to image the tumors. Theeffectiveness of the contrast agents can be readily seen by comparisonto the images obtain from animals that are not administered a contrastagent.

[0692] This model can also be used to assess the compounds of thepresent invention comprised of heavy atoms as X-ray contrast agents.After administration of the appropriate amount of the X-ray absorbingcompounds, the whole animal can be placed in a commercially availableX-ray imager to image the tumors. The effectiveness of the contrastagents can be readily seen by comparison to the images obtain fromanimals that are not administered a contrast agent.

[0693] This model can also be used to assess the compounds of thepresent invention comprised of an echogenic gas containing surfactantmicrosphere as ultrasound contrast agents. After administration of theappropriate amount of the echogenic compounds, the tumors in the animalcan be imaging using an ultrasound probe held proximate to the tumors.The effectiveness of the contrast agents can be readily seen bycomparison to the images obtain from animals that are not administered acontrast agent.

[0694] Rabbit Matrigel Model

[0695] This model was adapted from a matrigel model intended for thestudy of angiogenesis in mice. Matrigel (Becton & Dickinson, USA) is abasement membrane rich in laminin, collagen IV, entactin, HSPG and othergrowth factors. When combined with growth factors such as bFGF [500ng/ml] or VEGF [2 μg/ml] and injected subcutaneously into themid-abdominal region of the mice, it solidifies into a gel andstimulates angiogenesis at the site of injection within 4-8 days. In therabbit model, New Zealand White rabbits (2.5-3.0 kg) are injected with2.0 ml of matrigel, plus 1 μg bFGF and 4 μg VEGF. Theradiopharmaceutical is then injected 7 days later and the imagesobtained.

[0696] This model can also be used to assess the effectiveness of theradiopharmaceuticals of the present invention comprised of a beta, alphaor Auger electron emitting isotope. The radiopharmaceuticals areadministered in appropriate amounts and the uptake at the angiogenicsites can be quantified either non-invasively by imaging for thoseisotopes with a coincident imageable gamma emission, or by excision ofthe angiogenic sites and counting the amount of radioactivity present bystandard techniques. The therapeutic effect of the radiopharmaceuticalscan be assessed by monitoring the rate of growth of the angiogenic sitesin control rabbits versus those in the rabbits administered theradiopharmaceuticals of the present invention.

[0697] This model can also be used to assess the compounds of thepresent invention comprised of paramagnetic metals as MRI contrastagents. After administration of the appropriate amount of theparamagnetic compounds, the whole animal can be placed in a commerciallyavailable magnetic resonance imager to image the angiogenic sites. Theeffectiveness of the contrast agents can be readily seen by comparisonto the images obtain from animals that are not administered a contrastagent.

[0698] This model can also be used to assess the compounds of thepresent invention comprised of heavy atoms as X-ray contrast agents.After administration of the appropriate amount of the X-ray absorbingcompounds, the whole animal can be placed in a commercially availableX-ray imager to image the angiogenic sites. The effectiveness of thecontrast agents can be readily seen by comparison to the images obtainfrom animals that are not administered a contrast agent.

[0699] This model can also be used to assess the compounds of thepresent invention comprised of an echogenic gas containing surfactantmicrosphere as ultrasound contrast agents. After administration of theappropriate amount of the echogenic compounds, the angiogenic sites inthe animal can be imaging using an ultrasound probe held proximate tothe tumors. The effectiveness of the contrast agents can be readily seenby comparison to the images obtain from animals that are notadministered a contrast agent.

[0700] Canine Spontaneous Tumor Model

[0701] Adult dogs with spontaneous mammary tumors were sedated withxylazine (20 mg/kg)/atropine (1 ml/kg). Upon sedation the animals wereintubated using ketamine (5 mg/kg)/diazepam (0.25 mg/kg) for fullanethesia. Chemical restraint was continued with ketamine (3mg/kg)/xylazine (6 mg/kg) titrating as necessary. If required theanimals were ventilated with room air via an endotrachael tube (12strokes/min, 25 ml/kg) during the study. Peripheral veins werecatheterized using 20G I.V. catheters, one to serve as an infusion portfor compound while the other for exfusion of blood samples. Heart rateand EKG were monitored using a cardiotachometer (Biotech, Grass Quincy,Mass.) triggered from a lead II electrocardiogram generated by limbleads. Blood samples are generally taken at ˜10 minutes (control), endof infusion, (1 minute), 15 min, 30 min, 60 min, 90 min, and 120 min forwhole blood cell number and counting. Radiopharmaceutical dose was 300μCi/kg adminitered as an i.v. bolus with saline flush. Parameters weremonitored continuously on a polygraph recorder (Model 7E Grass) at apaper speed of 10 mm/min or 10 mm/sec.

[0702] Imaging of the laterals were for 2 hours with a 256×256 matrix,no zoom, 5 minute dynamic images. A known source is placed in the imagefield (20-90 μCi) to evaluate region of interest (ROI) uptake. Imageswere also acquired 24 hours post injection to determine retention of thecompound in the tumor. The uptake is determined by taking the fractionof the total counts in an inscribed area for ROI/source and multiplyingthe known μCi. The result is μCi for the ROI.

[0703] This model can also be used to assess the effectiveness of theradiopharmaceuticals of the present invention comprised of a beta, alphaor Auger electron emitting isotope. The radiopharmaceuticals areadministered in appropriate amounts and the uptake in the tumors can bequantified either non-invasively by imaging for those isotopes with acoincident imageable gamma emission, or by excision of the tumors andcounting the amount of radioactivity present by standard techniques. Thetherapeutic effect of the radiopharmaceuticals can be assessed bymonitoring the size of the tumors over time.

[0704] This model can also be used to assess the compounds of thepresent invention comprised of paramagnetic metals as MRI contrastagents. After administration of the appropriate amount of theparamagnetic compounds, the whole animal can be placed in a commerciallyavailable magnetic resonance imager to image the tumors. Theeffectiveness of the contrast agents can be readily seen by comparisonto the images obtain from animals that are not administered a contrastagent.

[0705] This model can also be used to assess the compounds of thepresent invention comprised of heavy atoms as X-ray contrast agents.After administration of the appropriate amount of the X-ray absorbingcompounds, the whole animal can be placed in a commercially availableX-ray imager to image the tumors. The effectiveness of the contrastagents can be readily seen by comparison to the images obtain fromanimals that are not administered a contrast agent.

[0706] This model can also be used to assess the compounds of thepresent invention comprised of an echogenic gas containing surfactantmicrosphere as ultrasound contrast agents. After administration of theappropriate amount of the echogenic compounds, the tumors in the animalcan be imaging using an ultrasound probe held proximate to the tumors.The effectiveness of the contrast agents can be readily seen bycomparison to the images obtain from animals that are not administered acontrast agent.

[0707] Cardiovascular disease models that can be used to assess thediagnostic radiopharmaceuticals, magnetic resonance, X-ray andultrasound contrast agents of the present invention are reviewed in J.Nucl. Cardiol., 1998, 5, 167-83. There are several well establishedrabbit models of atherosclerosis; one model produces predominantlyproliferating smooth muscle cells by balloon deendothelialization ofinfradiaphragmatic abdominal aorta to simulate restenotic lesions;another model that produces simulated advanced human atheroscleroticplaque by balloon deendothelialization followed by a high cholesteroldiet.

[0708] A model of congestive heart failure is described in Am. J.Physiol., 1998, 274, H1516-23. In general, Yorkshire pigs are randomlyassigned to undergo 3 wks of rapid atrial pacing at 240 beats/min. or tobe sham controls. The pigs are chronically instrumented to measure leftventricular function in the conscious state. The pigs are anesthetized.A shielded stimulating electrode is sutured onto the left atrium,connected to a modified programmable pace maker and buried in asubcutaneous pocket. The pericardium is closed loosely, the thoracotomyis closed, and the pleural space is evacuated of air. After a recoveryperiod of 7-10 days, the pacemaker is activated in the animals selectedto undergo chronic rapid pacing. The animals are sedated, the pacemakeris deactivated (pacing groups only. After a 30 min stabilization period,indexes of LV function and geometry are determined (by echocardiographyas a control) by injecting the radiolabeled compound. Forbiodistribution, the animals are anesthetized, the heart extirpate andthe LV apex and midventricular regions are evaluated.

[0709] A rat model of reversible coronary occlusion and reperfusion isdescribed in McNulty et al., J. Am. Physiol., 1996, H2283-9.

[0710] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise that as specifically describedherein.

What is claimed is described below:
 1. A compound, comprising: a targeting moiety and a chelator, wherein the targeting moiety is bound to the chelator, is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene nonpeptide, and binds to a receptor that is upregulated during angiogenesis and the compound has 0-1 linking groups between the targeting moiety and chelator.
 2. A compound according to claim 1, wherein the receptor is the integrin α_(v)β₃ or α_(v)β₅ and compound is of the formula: (Q)_(d)-L_(n)-C_(h) or (Q)_(d)-L_(n)-(C_(h))_(d′) wherein, Q is a compound of Formulae (Ia), (Ib) or (Ic):

wherein: R¹ and R³ are independently selected from the group: C₁-C₆ alkyl, benzyl, phenethyl, and a bond to L_(n); provided that one of R¹ and R³ is a bond to L_(n); R² is independently selected from the group: 2-benzimidazolylmethyl, 2-guanidinoethyl, 2-amino-2-pyridyl, 2-amino-2-pyridylmethyl, 5-amino-2-imidazolylmethyl, and 2-imidazolylmethyl; R⁴ is independently selected from H, C₁₋₆ alkyl or benzyl; R^(2a) is (CH₂)₃R^(3a); R^(3a) is selected from the group:

R^(4a) is independently selected from C₁₋₆ alkyl substituted with a bond to L_(n) or benzyl substituted with a bond to L_(n); R^(2b) is independently selected from the group:

the asterisks * denote optional positions for attaching L_(n); or Q is a peptide selected from the group:

R^(1p) is L-valine, D-valine or L-lysine optionally substituted on the ε amino group with a bond to L_(n); R^(2p) is L-phenylalanine, D-phenylalanine, D-1-naphthylalanine, 2-aminothiazole-4-acetic acid or tyrosine, the tyrosine optionally substituted on the hydroxy group with a bond to L_(n); R^(3p) is D-valine; R^(4p) is D-tyrosine substituted on the hydroxy group with a bond to L_(n); provided that one of R^(1p) and R^(2p) in each Q is substituted with a bond to L_(n), and further provided that when R^(2p) is 2-aminothiazole-4-acetic acid, K is N-methylarginine; provided that at least one Q is a compound of Formula Ia Ib, or Ic; d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; d′ is 1-100; L_(n) is a linking group having the formula: ((W)_(h)-(CR⁶R⁷)_(g))_(x)-(Z)_(k)-((CR^(6a)R^(7a))_(g′)-5215 (W)_(h′))_(x′); W is independently selected at each occurrence from the group: O, S, NH, NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)N R⁸, C(═O), C(═O)O, OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′); aa is independently at each occurrence an amino acid; Z is selected from the group: aryl substituted with 0-3 R¹⁰, C₃₋₁₀ cycloalkyl substituted with 0-3 R¹⁰, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R¹⁰; R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at each occurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkyl substituted with 0-3 R¹⁰, aryl substituted with 0-3 R¹⁰, benzyl substituted with 0-3 R¹⁰, and C₁-C₅ alkoxy substituted with 0-3 R¹⁰, NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to C_(h); R¹⁰ is independently selected at each occurrence from the group: a bond to C_(h), COOR¹¹, C(═O)NHR¹¹, NHC(═O)R¹¹, OH, NHR¹¹, SO₃H, PO₃H, —OPO₃H₂, —OSO₃H, aryl substituted with 0-3 R¹¹, C₁₋₅ alkyl substituted with 0-1 R¹², C₁₋₅ alkoxy substituted with 0-1 R¹², and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R¹¹; R¹¹ is independently selected at each occurrence from the group: H, alkyl substituted with 0-1 R¹², aryl substituted with 0-1 R¹², a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-1 R¹², C₃₋₁₀ cycloalkyl substituted with 0-1 R¹², polyalkylene glycol substituted with 0-1 R¹², carbohydrate substituted with 0-1 R¹², cyclodextrin substituted with 0-1 R¹², amino acid substituted with 0-1 R¹², polycarboxyalkyl substituted with 0-1 R¹², polyazaalkyl substituted with 0-1 R¹², peptide substituted with 0-1 R¹², wherein the peptide is comprised of 2-10 amino acids, 3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and a bond to C_(h); R¹² is a bond to C_(h); k is selected from 0, 1, and 2; h is selected from 0, 1, and 2; h′ is selected from 0, 1, and 2; g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; s′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; s″ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; x is selected from 0, 1, 2, 3, 4, and 5; x′ is selected from 0, 1, 2, 3, 4, and 5; C_(h) is a metal bonding unit having a formula selected from the group:

A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at each occurrence from the group: NR¹³, NR¹³R¹⁴, S, SH, S(Pg), O, OH, PR¹³, PR¹³R¹⁴, P(O)R¹⁵R¹⁶, and a bond to L_(n); E is a bond, CH, or a spacer group independently selected at each occurrence from the group: C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, aryl substituted with 0-3 R¹⁷, C₃₋₁₀ cycloalkyl substituted with 0-3 R¹⁷, heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, wherein the heterocyclo group is a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀ aryl-substituted with 0-3 R¹⁷, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R¹⁷; R¹³ and R¹⁴ are each independently selected from the group: a bond to L_(n), hydrogen, C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, aryl substituted with 0-3 R¹⁷, C₁₋₁₀ cycloalkyl substituted with 0-3 R¹⁷, heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, wherein the heterocyclo group is a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀ aryl-substituted with 0-3 R¹⁷, a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R¹⁷, and an electron, provided that when one of R¹³ or R¹⁴ is an electron, then the other is also an electron; alternatively, R¹³ and R¹⁴ combine to form ═C (R²⁰) (R²¹); R¹⁵ and R¹⁶ are each independently selected from the group: a bond to L_(n), —OH, C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, aryl substituted with 0-3 R¹⁷, C₃₋₁₀ cycloalkyl substituted with 0-3 R¹⁷, heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, wherein the heterocyclo group is a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀ aryl-substituted with 0-3 R¹⁷, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R¹⁷; R¹⁷ is independently selected at each occurrence from the group: a bond to L_(n), ═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R¹⁸, —C(═O)R¹⁸, —C(═O)N(R¹⁸)₂, —CHO, —CH₂OR¹⁸, —OC(═O)R¹⁸, —OC(═O)OR^(18a), —OR¹⁸, —OC(═O)N(R¹⁸)₂, —NR¹⁹C(═O)R¹⁸, —NR¹⁹C(═O)OR^(18a), —NR¹⁹C(═O)N(R¹⁸)₂, —NR¹⁹SO₂N(R¹⁸)₂, —NR¹⁹SO₂R^(18a), —SO₃H, —SO₂R^(18a), —SR¹⁸, —S(═O)R^(18a), —SO₂N(R¹⁸)₂, —N(R¹⁸)₂, —NHC(═S)NHR¹⁸, ═NOR¹⁸, NO₂, —C(═O)NHOR¹⁸, —C(═O)NHNR¹⁸R^(18a), —OCH₂CO₂H, 2-(1-morpholino)ethoxy, C₁-C₅ alkyl, C₂-C₄ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkylmethyl, C₂-C₆ alkoxyalkyl, aryl substituted with 0-2 R¹⁸, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O; R^(18,) R^(18a), and R¹⁹ are independently selected at each occurrence from the group: a bond to L_(n), H, C₁-C₆ alkyl, phenyl, benzyl, C₁-C₆ alkoxy, halide, nitro, cyano, and trifluoromethyl; Pg is a thiol protecting group; R²⁰ and R²¹ are independently selected from the group: H, C₁-C₁₀ alkyl, —CN, —CO₂R²⁵, —C(═O)R²⁵, —C(═O)N(R²⁵)₂, C₂-C₁₀ 1-alkene substituted with 0-3 R²³, C₂-C₁₀ 1-alkyne substituted with 0-3 R²³, aryl substituted with 0-3 R²³, unsaturated 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R²³, and unsaturated C₃₋₁₀ carbocycle substituted with 0-3 R²³; alternatively, R²⁰ and R²¹, taken together with the divalent carbon radical to which they are attached form:

R²² and R²³ are independently selected from the group: H, R²⁴, C₁-C₁₀ alkyl substituted with 0-3 R²⁴, C₂-C₁₀ alkenyl substituted with 0-3 R²⁴, C₂-C₁₀ alkynyl substituted with 0-3 R²⁴, aryl substituted with 0-3 R²⁴, a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R²⁴, and C₃₋₁₀ carbocycle substituted with 0-3 R²⁴; alternatively, R²², R²³ taken together form a fused aromatic or a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O; a and b indicate the positions of optional double bonds and n is 0 or 1; R²⁴ is independently selected at each occurrence from the group: ═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R²⁵, —C(═O)R²⁵, —C(═O)N(R²⁵)₂, —N(R²⁵)₃ ⁺, —CH₂OR²⁵, —OC(═O)R²⁵, —OC(═O)OR^(25a), —OR²⁵, —OC(═O)N(R²⁵)₂, —NR²⁶C(═O)R²⁵, —NR²⁶C(═O)OR^(25a), —NR²⁶C(═O)N(R²⁵)₂, —NR²⁶SO₂N(R²⁵)₂, —NR²⁶SO₂R^(25a), —SO₃H, —SO₂R^(25a), —SR²⁵, —S(═O)R^(25a), —SO₂N(R²⁵)₂, —N(R²⁵)₂, ═NOR²⁵, —C(═O)NHOR²⁵, —OCH₂CO₂H, and 2-(1-morpholino)ethoxy; and, R²⁵, R^(25a), and R²⁶ are each independently selected at each occurrence from the group: hydrogen and C₁-C₆ alkyl; and a pharmaceutically acceptable salt thereof.
 3. A compound according to claim 2, wherein: d is selected from 1, 2, 3, 4, and 5; d′ is 1-50; W is independently selected at each occurrence from the group: O, NH, NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)N R⁸, C(═O), C(═O)O, OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′); aa is independently at each occurrence an amino acid; Z is selected from the group: aryl substituted with 0-1 R¹⁰, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁰, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-1 R¹⁰; R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at each occurrence from the group: H, ═O, COOH, SO₃H, C₁-C₅ alkyl substituted with 0-1 R¹⁰, aryl substituted with 0-1 R¹⁰, benzyl substituted with 0-1 R¹⁰, and C₁-C₅ alkoxy substituted with 0-1 R¹⁰, NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to C_(h); k is 0 or 1; s is selected from 0, 1, 2, 3, 4, and 5; s′ is selected from 0, 1, 2, 3, 4, and 5; s″ is selected from 0, 1, 2, 3, 4, and 5; t is selected from 0, 1, 2, 3, 4, and 5; A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at each occurrence from the group: NR¹³, NR¹³R¹⁴, S, SH, S(Pg), OH, and a bond to L_(n); E is a bond, CH, or a spacer group independently selected at each occurrence from the group: C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, aryl substituted with 0-3 R¹⁷, C₃₋₁₀ cycloalkyl substituted with 0-3 R¹⁷, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R¹⁷; R¹³, and R¹⁴ are each independently selected from the group: a bond to L_(n), hydrogen, C₁-C₁₀ alkyl substituted with 0-3 R¹⁷, aryl substituted with 0-3 R¹⁷, a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R¹⁷, and an electron, provided that when one of R¹³ or R¹⁴ is an electron, then the other is also an electron; alternatively, R¹³ and R¹⁴ combine to form ═C(R²⁰) (R²¹); R¹⁷ is independently selected at each occurrence from the group: a bond to L_(n), ═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R¹⁸, —C(═O)R¹⁸, —C(═O)N(R¹⁸)₂, —CH₂OR¹⁸, —OC(═O)R¹⁸, —OC(═O)OR^(18a), —OR¹⁸, —OC(═O)N(R¹⁸)₂, —NR¹⁹C(═O)R¹⁸, —NR¹⁹C(═O)OR^(18a), —NR¹⁹C(═O)N(R¹⁸)₂, —NR¹⁹SO₂N(R¹⁸)₂, —NR¹⁹SO₂R^(18a), —SO₃H, —SO₂R^(18a), —S(═O)R^(18a), —SO₂N(R¹⁸)₂, —N(R¹⁸)₂, —NHC(═S)NHR¹⁸, ═NOR¹⁸, —C(═O)NHNR¹⁸R^(18a), —OCH₂CO₂H, and 2-(1-morpholino)ethoxy; R¹⁸, R^(18a), and R¹⁹ are independently selected at each occurrence from the group: a bond to L_(n), H, and C₁-C₆ alkyl; R²⁰ and R²¹ are independently selected from the group: H, C₁-C₅ alkyl, —CO₂R²⁵, C₂-C₅ 1-alkene substituted with 0-3 R²³, C₂-C₅ 1-alkyne substituted with 0-3 R²³, aryl substituted with 0-3 R²³, and unsaturated 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R²³; alternatively, R²⁰ and R²¹, taken together with the divalent carbon radical to which they are attached form:

R²² and R²³ are independently selected from the group: H, and R²⁴; alternatively, R²², R²³ taken together form a fused aromatic or a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O; R²⁴ is independently selected at each occurrence from the group: —CO₂R²⁵, —C(═O)N(R²⁵)₂, —CH₂OR²⁵, —OC(═O)R²⁵, —OR²⁵, —SO₃H, —N(R²⁵)₂, and —OCH₂CO₂H; and, R²⁵ is independently selected at each occurrence from the group: H and C₁-C₃ alkyl.
 4. A compound according to claim 3, wherein: R^(4a) is benzyl substituted with a bond to L_(n);

A¹ is selected from the group: OH, and a bond to L_(n); A², A⁴, and A⁶ are each N; A³, A⁵, and A⁸ are each OH; A⁷ is a bond to L_(n) or NH-bond to L_(n); E is a C₂ alkyl substituted with 0-1 R¹⁷; R¹⁷ is ═O; alternatively, C_(h) is

A¹ is selected from the group: OH, and a bond to L_(n); A², A³ and A⁴ are each N; A⁵, A⁶ and A⁸ are each OH; A⁷ is a bond to L_(n); E is a C₂ alkyl substituted with 0-1 R¹⁷; R¹⁷ is ═O; alternatively, C_(h) is

A¹ is NH₂ or N═C(R²⁰)(R²¹); E is a bond; A² is NHR¹³; R¹³ is a heterocycle substituted with R¹⁷, the heterocycle being selected from pyridine and pyrimidine; R¹⁷ is selected from a bond to L_(n), C(═O)NHR¹⁸ and C(═O)R¹⁸; R¹⁸ is a bond to L_(n); R²⁴ is selected from the group: —CO₂R²⁵, —OR²⁵, —SO₃H, and —N(R²⁵)₂; and, R²⁵ is independently selected at each occurrence from the group: hydrogen and methyl.
 5. A compound according to claim 2, wherein the compound is selected from the group: (S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclodecyl)acetylamino)butanoyl amino)butanoic acid; (S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl) acetic acid; (S)-2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid; (S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl) benzenesulfonic acid; (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)butanoylamino) butanoic acid; (S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)propanoic acid; (S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4,4-dihydroxypentyl) carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino) butanoic acid; (S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)p ropyl)carbamoyl)-4-(2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclodecyl)acetylamino)-4-carboxybutanoylamino)-4-carboxybutanoylamino)butanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methyl carbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0] undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)butanoic acid; (S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino) propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid; (S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)propyl)carbamoyl) propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxy methyl)cyclododecyl)acetylamino) hexanoylamino)butanoic acid; (S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxy hexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl) acetylamino)butanoylamino)butanoylamino)hexyl)bicycl o[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid; (S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}[gamma-LysNH] carbamoyl)propyl)carbamoyl)-3-carboxypropyl) carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)butanoic acid; 4-[N-(3-{(2R)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H, 2H,5H-benzo[f]14-diazepin-4-yl)}propyl)carbamoyl] (4S)-4-[(4S)-4-(N-{(1S)-1-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo (1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-3-carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}butanoyl amino]butanoic acid; 2-(4-{3-[(6-{[(1E)-1-aza-2-(2-sulfophenyl)vinyl]amino}(3-pyridyl))carbonylamino]propyl} (2S)-7-{N-[2-(amidinoamino)ethyl]-N-methylcarbamoyl}-3-oxo-1H,2H,5H-benzo[f]1,4-diazepin-2-yl)acetic acid; and 2-[9-(N-{6-[(6-{[(1E)-1-aza-2-(2-sulfophenyl)vinyl]amino}(3-pyridyl))carbonylamino]hexyl}—N-(benzimidazol-2-ylmethyl)carbamoyl)(5S)-5,6,11-trihydro-dibenzo[b,e][7]annulen-5-yl]acetic acid; or a pharmaceutically acceptable salt form thereof.
 6. A kit comprising a compound of claim 2, or a pharmaceutically acceptable salt form thereof and a pharmaceutically acceptable carrier.
 7. A kit according to claim 6, wherein the kit further comprises one or more ancillary ligands and a reducing agent.
 8. A kit according to claim 7, wherein the ancillary ligands are tricine and TPPTS.
 9. A kit according to claim 7, wherein the reducing agent is tin(II).
 10. A diagnostic or therapeutic metallopharmaceutical composition, comprising: a metal, a chelator capable of chelating the metal and a targeting moiety, wherein the targeting moiety is bound to the chelator, is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene nonpeptide and binds to a receptor that is upregulated during angiogenesis and the compound has 0-1 linking groups between the targeting moiety and chelator.
 11. A composition according to claim 10, wherein the metallopharmaceutical is a diagnostic radiopharmaceutical, the metal is a radioisotope selected from the group: ^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, and ⁶⁸Ga, and the linking group is present between the targeting moiety and chelator.
 12. A composition according to claim 11, wherein the targeting moiety is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene and the receptor is α_(v)β₃ or α_(v)β₅.
 13. A composition according to claim 12, wherein the radioisotope is ^(99m)Tc or ⁹⁵Tc, the radiopharmaceutical further comprises a first ancillary ligand and a second ancillary ligand capable of stabilizing the radiopharmaceutical.
 14. A composition according to claim 13, wherein the radioisotope is ^(99m)Tc.
 15. A composition according to claim 14, wherein the radiopharmaceutical is selected from the group: ^(99m)Tc((S)-2-(2,5-diaza-5-(6((6-(diazenido)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl) acetic acid)(tricine)(TPPTS) and ^(99m)Tc((S)-2-(2,5-diaza-9-(N-(6-((6-(diazenido)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid)(tricine)(TPPTS);
 16. A composition according to claim 12, wherein the radioisotope is ¹¹¹In.
 17. A composition according to claim 16, wherein the radiopharmaceutical is selected from the group: ¹¹¹In complex of 6-(N-(3-(3-aza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-((2-((carboxymethyl)(2-((carboxymethyl)methylamino)ethyl)amino) ethyl)(2-((carboxymethyl)ethylamino)ethyl)amino)-acetylamino)-4-oxooctane-1,8-dicarboxylic acid; ¹¹¹In complex of (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)butanoylamino) butanoic acid; and ¹¹¹In complex of (S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)propanoic acid.
 18. A composition according to claim 10, wherein the metallopharmaceutical is a therapeutic radiopharmaceutical, the metal is a radioisotope selected from the group: ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir, and the linking group is present between the targeting moiety and chelator.
 19. A composition according to claim 18, wherein the targeting moiety is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene and the receptor is α_(v)β₃ or α_(v)β₅.
 20. A composition according to claim 19, wherein the radioisotope is ¹⁴⁹Pm.
 21. A composition according to claim 20, wherein the radiopharmaceutical is selected from the group: the Pm-149 complex of (S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclodecyl)acetylamino)butanoyl amino)butanoic acid; and the Pm-149 complex of (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)butanoylamino) butanoic acid.
 22. A composition according to claim 19, wherein the radioisotope is ¹⁷⁷Lu.
 23. A composition according to claim 22, wherein the radiopharmaceutical is selected from the group: the Lu-177 complex of (S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclodecyl)acetylamino)butanoyl amino)butanoic acid; and the Lu-177 complex of (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)butanoylamino) butanoic acid; and the Lu-177 complex of (S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)propanoic acid.
 24. A composition according to claim 19, wherein the radioisotope is ⁹⁰Y.
 25. A composition according to claim 24, wherein the radiopharmaceutical is selected from the group: the Y-90 complex of (S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclodecyl)acetylamino)butanoyl amino)butanoic acid; and the Y-90 complex of (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)butanoylamino) butanoic acid; and the Y-90 complex of (S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)propanoic acid.
 26. A composition according to claim 10, wherein the metallopharmaceutical is a MRI contrast agent, the metal is a paramagnetic metal ion selected from the group: Gd(III), Dy(III), Fe(III), and Mn(II), and the linking group is present between the targeting moiety and chelator.
 27. A composition according to claim 26, wherein the targeting moiety is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene and the receptor is α_(v)β₃ or α_(v)β₅.
 28. A composition according to claim 27, wherein the metal ion is Gd(III).
 29. A composition according to claim 10, wherein the metallopharmaceutical is a X-ray contrast agent, the metal is selected from the group: Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au, Yb, Dy, Cu, Rh, Ag, and Ir, and the linking group is present between the targeting moiety and chelator.
 30. A method of treating rheumatoid arthritis in a patient comprising: administering a therapeutic radiopharmaceutical of claim 18 capable of localizing in new angiogenic vasculature to a patient by injection or infusion.
 31. A method of treating cancer in a patient comprising: administering to a patient in need thereof a therapeutic radiopharmaceutical of claim 18 by injection or infusion.
 32. A method of treating restenosis in a patient comprising: administering to a patient, either systemically or locally, a therapeutic radiopharmaceutical of claim 18 capable of localizing in the restenotic area and delivering an effective dose of radiation.
 33. A method of imaging cancer in a patient comprising: (1) administering a diagnostic radiopharmaceutical of claim 11 to a patient by injection or infusion; (2) imaging the patient using planar or SPECT gamma scintigraphy, or positron emission tomography.
 34. A method of imaging cancer in a patient comprising: (1) administering a MRI contrast agent of claim 26; and (2) imaging the patient using magnetic resonance imaging.
 35. A method of imaging cancer in a patient comprising: (1) administering an X-ray contrast agent of claim 29; and (2) imaging the patient using X-ray computed tomography.
 36. A method of imaging therapeutic angiogenesis in a patient comprising: (1) administering a diagnostic radiopharmaceutical, a MRI contrast agent, or a X-ray contrast agent of claim 10 to a patient by injection or infusion; (2) imaging the area of the patient wherein the desired formation of new blood vessels is located.
 37. A method of imaging atherosclerosis in a patient comprising: (1) administering a diagnostic radiopharmaceutical, a MRI contrast agent, or a X-ray contrast agent of claim 10 to a patient by injection or infusion; (2) imaging the area of the patient wherein the atherosclerosis is located.
 38. A method of imaging restenosis in a patient comprising: (1) administering a diagnostic radiopharmaceutical, a MRI contrast agent, or a X-ray contrast agent of claim 10 to a patient by injection or infusion; (2) imaging the area of the patient wherein the restenosis is located.
 39. A method of imaging cardiac ischemia in a patient comprising: (1) administering a diagnostic radiopharmaceutical, a MRI contrast agent, or a X-ray contrast agent of claim 10 to a patient by injection or infusion; (2) imaging the area of the myocardium wherein the ischemic region is located.
 40. A method of imaging myocardial reperfusion injury in a patient comprising: (1) administering a diagnostic radiopharmaceutical, a MRI contrast agent, or a X-ray contrast agent of claim 10 to a patient by injection or infusion; (2) imaging the area of myocardium wherein the reperfusion injury is located.
 41. A compound, comprising: a targeting moiety and a surfactant, wherein the targeting moiety is bound to the surfactant, is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene nonpeptide, and binds to a receptor that is upregulated during angiogenesis and the compound has 0-1 linking groups between the targeting moiety and surfactant.
 42. A compound according to claim 41, wherein the receptor is the integrin α_(v)β₃ or α_(v)β₅ and the compound is of the formula: (Q)_(d)-L_(n)-S_(f) wherein, Q is a compound of Formulae (Ia), (Ib) or (Ic):

wherein: R¹ and R³ are independently selected from the group: C₁-C₆ alkyl, benzyl, phenethyl, and a bond to L_(n); provided that one of R¹ and R³ is a bond to L_(n); R² is independently selected from the group: 2-benzimidazolylmethyl, 2-guanidinoethyl, 2-amino-2-pyridyl, 2-amino-2-pyridylmethyl, 5-amino-2-imidazolylmethyl, and 2-imidazolylmethyl; R⁴ is independently selected from H, C₁₋₆ alkyl or benzyl; R^(2a) is (CH₂)₃R^(3a); R^(3a) is selected from the group:

R^(4a) is independently selected from C₁₋₆ alkyl substituted with a bond to L_(n) or benzyl substituted with a bond to L_(n); R^(2b) is independently selected from the group:

the asterisks * denote optional positions for attaching L_(n); or Q is a peptide selected from the group:

R^(1p) is L-valine, D-valine or L-lysine optionally substituted on the ε amino group with a bond to L_(n); R^(2p) is L-phenylalanine, D-phenylalanine, D-1-naphthylalanine, 2-aminothiazole-4-acetic acid or tyrosine, the tyrosine optionally substituted on the hydroxy group with a bond to L_(n); R^(3p) is D-valine; R^(4p) is D-tyrosine substituted on the hydroxy group with a bond to L_(n); provided that one of R^(1p) and R^(2p) in each Q is substituted with a bond to L_(n), and further provided that when R^(2p) is 2-aminothiazole-4-acetic acid, K is N-methylarginine; provided that at least one Q is a compound of Formula Ia Ib, or Ic; d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; L_(n) is a linking group having the formula: ((W)_(h)-(CR⁶R⁷)_(g))_(x)-(Z)_(k)-((CR^(6a)R^(7a))_(g′)-(W)_(h′))_(x′); W is independently selected at each occurrence from the group: O, S, NH, NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)N R⁸, C(═O), C(═O)O, OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, (OCH₂CH₂)₂₀₋₂₀₀, (CH₂CH₂O)₂₀₋₂₀₀, (OCH₂CH₂CH₂)₂₀₋₂₀₀, (CH₂CH₂CH₂O)₂₀₋₂₀₀, and (aa)_(t′); aa is independently at each occurrence an amino acid; Z is selected from the group: aryl substituted with 0-3 R¹⁰, C₃₋₁₀ cycloalkyl substituted with 0-3 R¹⁰, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R¹⁰; R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at each occurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkyl substituted with 0-3 R¹⁰, aryl substituted with 0-3 R¹⁰, benzyl substituted with 0-3 R¹⁰, and C₁-C₅ alkoxy substituted with 0-3 R¹⁰, NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to S_(f); R¹⁰ is independently selected at each occurrence from the group: a bond to S_(f), COOR¹¹, C(═O)NHR¹¹, NHC(═O)R¹¹, OH, NHR¹¹, SO₃H, PO₃H, —OPO₃H₂, —OSO₃H, aryl substituted with 0-3 R¹¹, C₁₋₅ alkyl substituted with 0-1 R¹², C₁₋₅ alkoxy substituted with 0-1 R¹², and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R¹¹; R¹¹ is independently selected at each occurrence from the group: H, alkyl substituted with 0-1 R¹², aryl substituted with 0-1 R¹², a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-1 R¹², C₃₋₁₀ cycloalkyl substituted with 0-1 R¹² 5934 , and a bond to S_(f); R¹² is a bond to S_(f); k is selected from 0, 1, and 2; h is selected from 0, 1, and 2; h′ is selected from 0, 1, and 2; g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; x is selected from 0, 1, 2, 3, 4, and 5; x′ is selected from 0, 1, 2, 3, 4, and 5; S_(f) is a surfactant which is a lipid or a compound of the formula:

A⁹ is selected from the group: OH and OR²⁷; A¹⁰ is OR²⁷; R²⁷ is C(═O)C₁₋₂₀ alkyl; E¹ is C₁₋₁₀ alkylene substituted with 1-3 R²⁸; R²⁸ is independently selected at each occurrence from the group: R³⁰, —PO₃H—R³⁰, ═O, —CO₂R²⁹, —C(═O)R²⁹, —C(═O)N(R²⁹)₂, —CH₂OR²⁹, —OR²⁹, —N(R²⁹)₂, C₁-C₅ alkyl, and C₂-C₄ alkenyl; R²⁹ is independently selected at each occurrence from the group: R³⁰, H, C₁-C₆ alkyl, phenyl, benzyl, and trifluoromethyl; R³⁰ is a bond to L_(n); and a pharmaceutically acceptable salt thereof.
 43. A compound according to claim 42, wherein the compound is of the formula: Q-L_(n)-S_(f) wherein: Q is a compound of Formulae (Ia), (Ib), or (Ic):

R^(4a) is benzyl substituted with a bond to L_(n);

Z is selected from the group: aryl substituted with 0-1 R¹⁰, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁰, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-1 R¹⁰; R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at each occurrence from the group: H, ═O, COOH, SO₃H, C_(l)-C₅ alkyl substituted with 0-1 R¹⁰, aryl substituted with 0-1 R¹⁰, benzyl substituted with 0-1 R¹⁰, and C₁-C₅ alkoxy substituted with 0-1 R¹⁰, NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to S_(f); k is 0 or 1; S_(f) is a surfactant which is a lipid or a compound of the formula:

A⁹ is OR²⁷; A¹⁰ is OR²⁷; R²⁷ is C(═O)C₁₋₁₅ alkyl; E¹ is C₁₋₄ alkylene substituted with 1-3 R²⁸; R²⁸ is independently selected at each occurrence from the group: R³⁰, —PO₃H—R³⁰, ═O, —CO₂R²⁹, —C(═O)R²⁹, —CH₂OR²⁹, —OR²⁹, and C₁-C₅ alkyl; R²⁹ is independently selected at each occurrence from the group: R³⁰, H, C₁-C₆ alkyl, phenyl, and benzyl; R³⁰ is a bond to L_(n); and a pharmaceutically acceptable salt thereof.
 44. A compound according to claim 43, wherein the compound selected from the group: Sodium 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid-dodecoanoate conjugate; DPPE-PEG₃₄₀₀-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]updeca-1(7),8,10-trien-3-yl)acetic acid]-dodecoanoate conjugate; and [(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)carbamoyl)propyl)carbamoyl]-w-amino-PEG₃₄₀₀-dodecanoate-DPPE conjugate.
 45. An ultrasound contrast agent composition, comprising: (a) a compound of claim 41, comprising: a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene that binds to the integrin α_(v)β₃, or α_(v)β₅, a surfactant and a linking group between the benzodiazepine and the surfactant; (b) a parenterally acceptable carrier; and, (c) an echogenic gas.
 46. An ultrasound contrast agent composition of claim 45, further comprising: 1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, and N-(methoxypolyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.
 47. An ultrasound contrast agent composition of claim 46, wherein the echogenic gas is a C₂₋₅ perfluorocarbon.
 48. A method of imaging cancer in a patient comprising: (1) administering, by injection or infusion, an ultrasound contrast agent composition of claim 45 to a patient; and (2) imaging the patient using sonography.
 49. A method of imaging therapeutic angiogenesis in a patient comprising: (1) administering, by injection or infusion, an ultrasound contrast agent composition of claim 45 to a patient; (2) imaging the area of the patient wherein the desired formation of new blood vessels is located.
 50. A method of imaging atherosclerosis in a patient comprising: (1) administering, by injection or infusion, an ultrasound contrast agent composition of claim 45 to a patient; (2) imaging the area of the patient wherein the atherosclerosis is located.
 51. A method of imaging restenosis in a patient comprising: (1) administering, by injection or infusion, an ultrasound contrast agent composition of claim 45 to a patient; (2) imaging the area of the patient wherein the restenosis is located.
 52. A method of imaging cardiac ischemia in a patient comprising: (1) administering, by injection or infusion, an ultrasound contrast agent composition of claim 45 to a patient; (2) imaging the area of the myocardium wherein the ischemic region is located.
 53. A method of imaging myocardial reperfusion injury in a patient comprising: (1) administering, by injection or infusion, an ultrasound contrast agent composition of claim 45 to a patient; (2) imaging the area of myocardium wherein the reperfusion injury is located.
 54. A therapeutic radiopharmaceutical composition, comprising: (a) a therapeutic radiopharmaceutical of claim 19; and, (b) a parenterally acceptable carrier.
 55. A diagnostic radiopharmaceutical composition, comprising: (a) a diagnostic radiopharmaceutical, a MRI contrast agent, or a X-ray contrast agent of claim 10; and, (b) a parenterally acceptable carrier. 